Author's personal copy

Pulmonary delivery of therapeutic siRNA

Jenny Ka-Wing Lam a,⁎, Wanling Liang a, Hak-Kim Chan b

a Department of Pharmacology & Pharmacy, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kongb Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia

a b s t r a c ta r t i c l e i n f o

Article history:Received 2 February 2011Accepted 19 February 2011Available online 26 February 2011

Keywords:siRNA deliveryLungInhalationAerosolNon-viral

Small interfering RNA (siRNA) has a huge potential for the treatment or prevention of various lungdiseases. Oncethe RNA molecules have successfully entered the target cells, they could inhibit the expression of specific genesequence through RNA interference (RNAi)mechanism and generate therapeutic effects. The biggest obstacle totranslating siRNA therapy from the laboratories into the clinics is delivery. An ideal delivery agent should protectthe siRNA from enzymatic degradation, facilitate cellular uptake and promote endosomal escape inside the cells,with negligible toxicity. Lung targeting could be achieved by systemic delivery or pulmonary delivery. The latterroute of administration could potentially enhance siRNA retention in the lungs and reduce systemic toxic effects.However the presence ofmucus, themucociliary clearance actions and the high degree branching of the airwayspresent major barriers to targeted pulmonary delivery. The delivery systems need to be designed carefully inorder to maximize the siRNA deposition to the diseased area of the airways. In most of the pulmonary siRNAtherapy studies in vivo, siRNAwas delivered either intratracheally or intranasally. Very limitedworkwas done onthe formulation of siRNA for inhalationwhich is believed to be the direction for future development. This reviewfocuses on the latest development of pulmonary delivery of siRNA for the treatment of various lung diseases.

© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Delivering siRNA to the lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1. Challenges of pulmonary delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.2. Intracellular barriers to siRNA delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3. Pulmonary route of administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.1. Inhalation route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.2. Intratracheal route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.3. Intranasal route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. Non-viral delivery of siRNA to the lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.1. Naked siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.2. Lipid-based delivery vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

4.2.1. Cationic lipoplexes and liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.2.2. PEGylated lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.2.3. Neutral lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.2.4. Lipids particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.2.5. Lipid-like molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

4.3. Polymer-based delivery vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.3.1. PEI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.3.2. Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.3.3. PLGA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4.4. Peptide-based delivery vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Advanced Drug Delivery Reviews 64 (2012) 1–15

⁎ Corresponding author. Tel.: +852 28199599; fax: +852 28170859.E-mail address: jkwlam@hku.hk (J.K.W. Lam).

0169-409X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.addr.2011.02.006

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

j ourna l homepage: www.e lsev ie r.com/ locate /addr

Author's personal copy

1. Introduction

RNA interference (RNAi) has huge therapeutic potential in treatingmany diseases by silencing the expression of the target gene in a post-transcriptional manner. The mechanism of RNAi has been extensivelyreviewed in the literature [1–6]. In brief, RNAi can be achievedartificially by three major ways: (i) introducing long double strandedRNA (dsRNA) which is cleaved into small interfering RNA (siRNA) bythe enzyme Dicer in the cytoplasm, leading to the degradation oftarget mRNA; (ii) introducing plasmid DNA encodes for short hairpinRNA (shRNA) which is processed by Dicer into siRNA; and (iii)introducing small interfering RNA (siRNA) directly to initiate themRNA degradation process.

For the last two decades, scientists have been investigating the useof nucleic acids including DNA and antisense oligonucleotide astherapeutic agents. The biggest hurdle we are encountering isdelivery, and the same problem applies to RNAi therapy. Beingnegatively charged hydrophilic macromolecules that are highlysusceptible to nuclease degradation, nucleic acids are incapable ofcrossing the biological membrane on their own to reach the site ofaction. A delivery vector is therefore required to protect thetherapeutic nucleic acids from enzymatic degradation, facilitatecellular uptake and release nucleic acids at the site of action insidethe cells. Amongst the types of nucleic acids that are involved in theRNAi, siRNA is the most popular candidate being studied in RNAitherapy. Introduction of long dsRNA (typically consist of 500 to1000 base pairs) is known to induce interferon (IFN) response inmammalian cells [7,8], rendering it unsuitable for RNAi therapy,whereas siRNA (typically consist of 21–23 base pairs) can avoid theINF response. From the delivery perspective, siRNA has advantageover the plasmid DNA encoding shRNA. In order for DNA to beproperly expressed, it must enter the nucleus where transcriptiontakes place. Nuclear entry is an extremely inefficient process and thisis indeed considered to be one of the biggest barriers to the success ofgene therapy. On the other hand, siRNA targets the RNA-inducedsilencing complex (RISC), which is located in the cytoplasm. AlthoughshRNA has a higher gene silencing potency than siRNA [9,10], therelatively ease of delivery makes siRNA a better candidate for RNAitherapy. In fact, delivery of siRNA has more success than other RNAimolecules. In 2004, the first human clinical trial of RNAi therapy wasinitiated for the treatment of age-related macular degeneration(AMD) with siRNA targeting VEGF-receptor 1 delivered intravitreally[11,12]. Since then a number of clinical trials of siRNA therapy arebeing conducted for different conditions [13], including solid tumor

cancers [14] and respiratory syncytial virus (RSV) infection [15,16].Table 1 shows the summary of clinical trials of siRNA therapy.

RNAi can be potentially used to treat or prevent diseases affectingthe airways, such as lung cancer [26–30], various types of respiratoryinfectious diseases [16,31–36], airway inflammatory diseases [37–39]and cystic fibrosis [40]. siRNA delivery to the lungs could be achievedeither by local delivery or systemic delivery. The former route offersseveral important benefits over the latter, such as a lower dose ofsiRNA is required, reduction of undesirable systemic side effects andimproved siRNA stability due to lower nuclease activity in the airwaysthan in the serum. Furthermore lungs could potentially serve as aninteresting site for delivering siRNA for systemic effect due to itshigh vascularization, large surface area and ultra thin epithelium ofthe alveoli [41]. All these factors allow efficient and rapid siRNAabsorption.

The process of pulmonary siRNA delivery is summarized in Fig. 1.There are several ways to administer siRNA locally into the lungs.Inhalation is the most common and the easiest method for pulmonarydrug delivery and could be applied to siRNA delivery. siRNA forinhalation can be formulated into liquid aerosol or dry powderaerosol. The intranasal route is another common way to deliver siRNAinto the airways due to the ease of administration and the intranasalsiRNA preparation can be easily administered into the nasal cavity asnasal suspension. Intratracheal route of administration is alsoemployed to deliver siRNA into the lungs. However this method ofadministration is relatively invasive and non-physiologic [42]. It isgenerally considered to be used in animal studies only rather than forclinical applications. Regardless of the administration route, it is veryimportant to maintain siRNA stability and biological activity duringmanufacturing and delivery.

Similar to DNA delivery, both viral and non-viral vectors are beingemployed to deliver siRNA. Viruses are extremely efficient nucleicacids delivery vectors as they are evolved to transfer their geneticmaterial into the host cells. To enable viruses to deliver nucleic acidsfor therapeutic use, the viruses must first be genetically engineered toremove their virulence. The main advantage of using viral vectors istheir high transduction efficiency compared to transfection by non-viral methods. Viruses such as adenovirus [43–45], adeno-associatedvirus [46], and lentivirus [47,48] are being investigated to deliversiRNA to lung cells. Despite their high nucleic acids transfer efficiency,there are safety concerns regarding the use of viral vectors.

From the lessons we have learnt in DNA delivery, there are highrisks associated with the utilization of viral vectors [49]. Immuneresponse to viruses is the major challenge to viral delivery [50]. It

Table 1Summary of clinical trials of siRNA therapy.

Latest stage development Target disease Route of administration/delivery agent Company Product name Ref

III (terminated-unlikely tomeet primary endpoint)

AMD Intravitreal injection/naked siRNA Opko Health Bevasiranib(formerly Cand5)

[17]

II AMD Intravitreal injection/naked siRNA Allergen & SirnaTherapeutics

AGN211745(formerly Sirna-027)

[11]

II RSV infection Nasal spray/naked siRNA Alnylam Pharmaceuticals ALN-RSV01 [15,16]II Acute kidney injury Intravenous injection/naked siRNA Quark Pharmaceuticals QPI-1002 (formerly

I5NP)[18]

II AMD Intravitreal injection/siRNA Quark Pharmaceuticals,Pfizer

PF-4523665 (formerlyREDD14NP & RTP801i)

I/II Ocular hypertension & glaucoma Ophthalmic drops/naked siRNA Sylentis SYL040012I Solid state tumors Intravenous injection/cyclodextrin

nanoparticlesCalando Pharmaceuticals CALAA01 [14]

I Solid cancers with liverinvolvement

Intravenous injection/lipid nanoparticles Alnylam Pharmaceuticals ALN-VSP02 [19]

I Transthyretin mediatedamyloidasis (ATTR)

Intravenous injection/lipid nanoparticles Alnylam Pharmaceuticals ALN-TTR01 [20,21]

I Pachyonychia Congenita Intradermal injection/naked siRNA TransDerm TD101 [22,23]I Chronic optic nerve atrophy

& recent onset NAIONIntravitreal injection/naked siRNA Quark Pharmaceuticals QPI-1007

I Advanced solid cancer Intravenous infusion/liposomal nanoparticles Silence Therapeutics Atu027 [24,25]

AMD: Age-related Macular Degeneration, DME: Diabetic Macular Edema, NAION: Non-Arteritic Anterior Ischemic Optic Neuropathy, RSV: Respiratory Syncytical Virus.

2 J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

limits the effectiveness of therapy in repeated administration. Severecase of immune response can even lead to organ failure and theconsequences could be fatal [51]. Furthermore, several types of virusmay insert their genome randomly into the host chromosomes,disturbing gene function and resulting in insertional mutagenesis[52–54]. After the infamous Gelsinger case (18 years old volunteerGelsinger died in a gene therapy trial using adenovirus) [51] and theFrench X-SCID case (one third of children developed leukemia in agene therapy trial using retrovirus) [55], it is not hard to imagine thatthe chance of using viral vectors in any type of human nucleic acidstherapy to get approval from the FDAwill be extremely slim. The viralvectors are excellent agents to deliver siRNA for proof-of-conceptstudies, but appear to lack of real clinical application because of thesafety issues. Alternativeway to deliver siRNA is by non-viral methodswhich include any method that does not involve the use of viruses.The therapeutic siRNA are either administered directly into the site ofaction or delivered by non-viral vectors. Commonly used non-viralvectors for siRNA delivery include lipids, polymers and peptides.

Thomas et al. reviewed the non-viral siRNA delivery to the lung[56] and the review (published in 2007) focused on the delivery ofsiRNA for the treatment of three different lung virus infections:influenza, respiratory syncytial virus infection and severe acuterespiratory syndrome (SARS). The field of siRNA delivery has rapidlyexpanded since then and a good number of in vivo studies werecarried out in the past few years to investigate the local delivery ofsiRNA against various lung diseases including lung cancer [57],mycobacteria infection [36], pulmonary fibrosis [58,59], respiratoryalphaherpesvirus infection [31], and other endogenous gene targets inthe lungs [60,61]. In this review, we present the latest development ofsiRNA therapy for the treatment of various lung diseases. We discuss

the challenges to pulmonary drug administration and barriers tosiRNA delivery to the lung, including both extracellular and intra-cellular barriers, with the focus on non-viral delivery methods. Thefindings of recent clinical studies of pulmonary siRNA therapy are alsodiscussed.

2. Delivering siRNA to the lung

2.1. Challenges of pulmonary delivery

The pulmonary delivery of therapeutic macromolecules such asproteins and peptides has been investigated for over thirty years [41].The challenges of siRNA delivery via the pulmonary route are similarto the delivery of other macromolecules. In order to develop anefficient siRNA pulmonary delivery system, it is important to under-stand the anatomical and physiological properties of the respiratorytract in the first place.

The respiratory tract can be divided into two regions: (i) theconducting region which consists of nasal cavity, pharynx, trachea,bronchi and bronchioles; and (ii) the respiratory region whichconsists of the respiratory bronchioles and alveoli. The conductingregion is responsible for air conductance and the respiratory region iswhere the gaseous exchange takes place. The most prominent featureof the respiratory tract is the high degree of branching. According tothe Wiebel's model of lung, there are 24 generations in total. Thishighly branched structure comprises airways with varying length anddiameter presents an early barrier to targeted pulmonary delivery.Many lung diseases affect the lower region of the lungs. For thetherapeutic agents to reach the diseased area, they must follow theairstream around the bend along the branched airway to the deep

Fig. 1. Schematic illustrates steps involved in the delivery of siRNA into the lungs.

3J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

lung area. The size of particles is an important factor in determiningthe site of deposition as illustrated in Fig. 2 [62]. In pulmonarydelivery, size of particle is expressed in terms of aerodynamicdiameter. Large particles (N6 μm aerodynamic diameter) carry highmomentum and are more likely to be impacted on the airway wall atbifurcations instead of following the changing airstream. Thereforethey are usually deposited higher up in the airway such as the back ofthe throat or pharynx. For small particles (b1 μm aerodynamicdiameter), their movements are determined by Brownian motion.They are mostly exhaled during normal tidal breathing but pausingcan enhance their deposition as the probability of the latter isproportional to the square root of time [63]. The optimal particle sizefor efficient deposition at the lower respiratory tract is found to bebetween 1 and 5 μm [41,64]. As the particle size further decreasestowards the nanoscale, deposition in the lung increases again due tothe increasing diffusional mobility [65]. For nanoparticles that are lessthan 100 nm, they appear to settle effectively to the alveolar regionwith a fractional deposition of around 50%. However, these ultrafineparticles usually enter the lungs as larger agglomerates which can bebroken down relatively easily into smaller particles on deposition.Various elimination pathways for nanoparticles exist in the lungs,including coughing, dissolution, mucociliarly escalator, translocationfrom the airways to other sites, phagocytosis by macrophages andneuronal uptake. What has not been established is the quantitativerelationship of these pathways [66]. Very little is known about exactlyhow siRNA is cleared from the lungs.

Major barriers to pulmonary delivery include the mucociliaryclearance action of the ciliated epithelial cells, and the presence ofmucus, alveolar fluid and macrophages along different parts of theairways [67,68]. Particles that are deposited on the ciliated cells arerapidly removed by mucociliary clearance and are eventually beingcoughed up or swallowed. The mucus lines the respiratory epitheliumfrom the nasal cavity to the terminal bronchioles [69]. The majorcomponent of mucus is mucins which are glycosylated proteins.Mucus constitutes a physical barrier as it increases the viscosity of themoist surface of the lung epithelial cells, thereby reducing drugpenetration and diffusion rate. The alveolar fluid is found on thesurface of alveoli epithelium as a thin layer of pulmonary surfactantwhich comprises phospholipids and other surfactant proteins. It hasbeen reported that the pulmonary surfactant severely impeded thetransfection efficiency of lipid-based nucleic acids delivery system,but not polymer-based system [70–72]. The alveolar macrophageslocated in the alveoli rapidly engulf the foreign particles byphagocytosis as a defense mechanism [73]. The siRNA that is takenup into the macrophages are subsequently degraded inside the cells.

At disease state, the physiological conditions of the airways mightbe altered and pose a huge impact on the efficiency of the pulmonarydelivery system. During infection and inflammation, there is anincrease in mucus secretion and the mucociliary clearance is impaired[73,74]. The thickness, the viscosity and the composition of the mucuslayer depend on the pathological condition and vary betweenindividual [67]. It is important that these factors are being consideredduring the development of pulmonary delivery system for differenttherapeutic applications.

To overcome the anatomical and physiological barriers of thelungs, several delivery strategies can be incorporated. Besides usingparticles with small aerodynamic diameter suitable for deposition inthe lower airways, it has been reported that the use of large porousparticles can effectively avoid phagocytosis by the alveolar macro-phages and prolong retention time in the lungs [75–77]. Porousparticles over 10 μm in geometric diameter usually have a smalleraerodynamic diameter so that they are within the ideal aerodynamicsize range for effective lung deposition, but their actual geometric sizeis too large to be removed by macrophages. To overcome the mucusbarrier, the use of mucolytic agents, such as nacystelyn which breaksdown the three dimensional gel network of mucus, or the use ofmucus inhibitor, such as glycopyrrolate which inhibit mucussecretion, could be considered [78]. However their clinical benefitsare limited [67,78]. Inhaled mannitol has been clinically proven toincrease the mucus clearance in patients with cystic fibrosis orbronchietasis [79,80] and to improve the hydration and surfaceproperties of sputum [81]. Reducing the mucus barrier by mannitolinhalation prior to the delivery of siRNA may thus be beneficial. Theuse of ultrasound and magnetic field has also been reported to directand control the site of deposition of nucleic acids delivery systems inthe airways [82–84].

2.2. Intracellular barriers to siRNA delivery

Once the siRNA has reached the surface of the target cells of therespiratory tract, assuming it successfully gets away with theextracellular barriers, it still has to cross the cellular membrane andgain access into the cytoplasm where RISC, the final target of siRNA, islocated. siRNA is a negatively charged hydrophilic macromoleculeswith a molecular weight around 13 kDa. Base on its physicochemicalproperties, siRNA is not able to cross the biological membrane on itsown. Therefore one of the main functions of a delivery vector is tofacilitate the cellular uptake of siRNA.

Endocytosis is the major cellular uptake pathway known to beinvolved in non-viral nucleic acids delivery [85]. For efficientendocytosis to occur, particles should be under 150 nm in size.Particles within this size range could also avoid macrophage uptakeand thereby delayed lung clearance [86]. For particles that enter therespiratory tract as large aggregates, they must be redispersed ordeaggregated into the appropriate size before endocytosis could takeplace. Yet there are several different types of endocytic pathwayswhich in turn affect the set of barriers the macromolecules mayencounter, and the eventual fate of molecules could be very different[85]. Clathrin-mediated endocytosis is the major and the best-characterized endocytic pathway that occurs constitutively in allmammalian cells. Particles that are taken up by the cells through thispathway are enclosed in clathrin-coated vesicles. They are thentransported into early endosomes, which fuse to form late endosomesand subsequently into the lysosomes. During the process, the pH insidethe vesicles drops gradually to as low as pH 5.0 in the lysosomeswheredegradative enzymes including nuclease are present. Therefore thetherapeutic siRNAmust be able to exit from the endosomes/lysosomesinto the cytoplasm before it is degraded by the nuclease (Fig. 1).

There are several strategies that can be employed to promoteendosomal escape of siRNA. Polymers such as polyethylenimine (PEI)have the ability to trigger endosomal release. The ‘proton sponge

Fig. 2. The effect of particle size on deposition in human respiratory tract following oralbreathing of unit-density spheres at the reference pattern: 15-s breathing-cycle periodand flow rate at 300 cm3 s!1.Adapted from Ref. [62].

4 J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

hypothesis’ suggested that due to the high buffering capacity of PEI overa wide range of pH, the polymer becomes protonated as the pH dropsinside the endosomes. This leads to the influx of chloride ions, protonsand subsequently water into the endosomes. Eventually high osmoticpressure develops, causing the endosomes to burst open and release thecontents into the cytoplasm [87]. However the cellular effect of‘endosomes bursting’ has not been properly discussed or evaluatedand the PEI is also known to be associated with toxicity problem.Another strategy is to incorporate pH sensitive fusogenic peptides to thedelivery system. Fusogenic peptides such as GALA [88], KALA [89,90]INF7 [91] etc., have been employed in siRNAdelivery both in vitro and invivo. These peptides have the ability to undergo pH-dependentconformational change. At low pH, they adopt a ‘membrane-disrupting’conformation and destabilize the endosomal membrane, therebyreleasing the contents of endosomes into the cytoplasm.

Apart from clathrin-mediated endocytosis, caveolae-mediatedendocytosis is another mechanism that is involved in the uptake ofnucleic acids delivery systems [85]. After internalization, the deliverysystems are enclosed in caveolin-coated vesicles called caveosomeswhich are non-acidic. Nuclease and other degradative enzymes areabsence in caveosomes. The delivery systems can be directlytransported to the Golgi and/or endoplasmic reticulum, therebyavoiding the lysosomal degradation. Since caveolin is abundantlyexpressed in many cell types including lung tissues [92,93], it is animportant route of cell internalization in pulmonary delivery, andperhaps a more efficient route compared to clathrin-mediatedendocytosis, especially with the delivery system that lacks the abilityto escape from the acidic endosomal compartments.

The entrapment of siRNA inside the endosomes/lysosomes notonly leads to the degradation of the nucleic acids, but may also triggerthe activation of innate immunity. There has been evidence suggestedthat synthetic siRNA can be recognized by the toll-like receptors(TLRs), TLR7 and TLR8, inside the endosomal compartments, therebystimulating the innate immune response [94–96]. 2′ modification ofsiRNA could circumvent the problem but this approachmay adverselyaffect the gene silencing efficiency. Alternatively, early escape from orcompletely bypassing the endosomes during the intracellular deliveryprocess could perhaps avoid this immunostimulatory activity ofsiRNA. Moschos et al. [61] reported that when TAT-conjugated siRNAwas administered to the lung of the mouse, no immune response wasobserved. However when penetratin-conjugated siRNA was admin-istered in the same way, innate immune response was observed,possibly through the activation of TLR. Since siRNA, TAT or penetratinalone did not induce innate immune responses, the different fatebetween TAT-conjugated and penetratin-conjugated siRNA systemscould only be explained by their different cellular uptake mechanism.This study demonstrated the importance of the cellular uptakepathway and the intracellular trafficking in establishing the safetyprofile as well as the efficiency of siRNA delivery.

Lastly, phagocytosis is another cellular uptake mechanism thatoccurs in the respiratory tract. Since this route of uptake can only beperformed by specialized cells such as alveolar macrophages, it is notexpected to play an important role in siRNA delivery [85]. Unless thetherapeutic siRNA is aimed to target the alveolar macrophages, e.g.treatment of mycobacterium infection, this pathway should be totallyavoided, as molecules being taken up this pathway are eventuallydegraded in the phagolysosomes of the cells.

3. Pulmonary route of administration

Direct pulmonary delivery in humans is achieved by inhalation ofaerosol generated by either an inhaler or nebulizer. Before a newtherapeutic agent is used in a human clinical trial, it must demonstratepre-clinical efficacy in suitable animal model with good translatabilityto humans [97]. There is no exception to therapeutic siRNA. In addi-tional to inhalation, intratracheal and intranasal route are often used

to deliver therapeutic agents including siRNA to the lungs of animalsbecause of the relatively simple setup. The summary of the in vivostudies of non-viral siRNA delivery to the lung is provided in Table 2,and the routes of administration commonly used in these studies areillustrated in Fig. 3. Intratracheal and intranasal methods may not bepractical in the clinical setting. Intratracheal route is an invasivemethod of delivery which is not appropriate for human use. Thesuccess in delivering siRNA via intranasal route in rodents which is anobligate nose breathers cannot be extrapolated to human use becauseof the very different lung anatomy [98]. It is therefore important totake into consideration the route of administration in animal studieswhen assessing the delivery and therapeutic efficacy of a formulationfor lung delivery.

3.1. Inhalation route

Inhalation is the most popular and a non-invasive way to delivertherapeutic agents into the lungs. Threemain types of inhalationdevicesare currently available to deliver drug to the lung through inhalation.These include metered dose inhalers (MDIs), dry powder inhalers(DPIs) and nebulizers.With appropriatemodification and optimization,these devices could be applied to pulmonary delivery of siRNA. To date,MDIs are the most commonly used inhalers. They are pressurizeddosage form in which the therapeutic agents are either dissolved orsuspended in propellants such as chlorofluorocarbons (CFCs) andhydrofluoroalkanes (HFAs). The propellants serve to provide a drivingpressure to aerosolize the drug for inhalation into the respiratory tract.However both CFCs andHFAs are known tohave environmental impact.The compatibility of formulation with propellants is another potentialproblemand the lung deposition is generally poorwithMDIs. Itmay notbe the best direction for developing inhalable siRNA.

DPIs are aerosol systems in which drugs are inhaled as clouds ofdry particles. The use of DPIs appears to be a promising way to deliversiRNA to the lungs as they demonstrated successful in vivo delivery ofother therapeutic macromolecules including insulin [108], parathy-roid hormone [109] and low molecular weight heparin [110,111].The formulation challenges and potential solutions for delivery ofmacromolecules such as proteins as powder aerosols have beenreviewed [112]. Formulating biological macromolecules as powdersfor aerosol delivery is a challenge as it requires not only flowabilityand dispersibility of the powders but also biochemical stability ofthe macromolecules. To satisfy the latter requirement, proteins areusually formulated in amorphous glasses which are, however, phy-sically unstable and tend to crystallize with inter-particulate bondformation and loss of powder dispersiblity. In addition, the biochem-ical stability requirement limits the manufacturing processes thatcan be used for protein powder production. Similar issues will beencountered for siRNA. Nevertheless, possible ways to tackle thesechallenges have been addressed [112]. The inhaled dry powder formof insulin (Exubera, marketed by Pfizer) was approved in Europe andthe US for the treatment of diabetes in 2006 [113]. Although theproduct was withdrawn from the market in the subsequent year,Pfizer stressed that disappointing sales is the major reason for thewithdrawal rather than safety or efficacy issues [114]. The safetyprofile of inhaled insulin was indeed reassuring and the efficacy wasnot inferior to the conventional injection formulations [115–119]. Asecond inhalable insulin product in dry powder form, Afresa, iscurrently awaiting for FDA approval. This encourages further deve-lopment of dry powder form of macromolecules for inhalation.

There are different designs of DPI device and their delivery per-formance may vary. The key advantages of DPIs are the improvedstability and sterility of biomolecules over liquid aerosols, and thepropellant-free formulation [120]. Inhalable dry powder forms ofproteins and peptides are commonly produced by spray-drying[121,122] and the same technique could be applied to siRNA[122,123]. Size of the spray-dried product must be carefully optimized

5J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

for efficient delivery to the desirable site along the respiratory tract. Asuitable delivery agent or formulation is required to protect thenucleic acids from degradation caused by the shear force and raisedtemperature during the drying process. The major drawback of DPIs isthat drug deposition could be dependent on the patient inspirationflow rate. Therefore a suitable DPI device must be carefully designedto minimize such variation. In addition, the problem associated withde-aggregation of dry powders must be overcome [120].

Nebulizers are used to generate liquid aerosol and can be utilizedto deliver large volumes of drug solutions or suspensions forinhalation. They are frequently used for drugs that are unsuitable tobe formulated into MDIs or DPIs, and could be considered for siRNAdelivery. During the process of nebulization, high shear stress isexerted on the siRNA which may lead to degradation of the nucleic

acids. This is a particular problem as 99% of generated aerosol dropletsare recycled back into the reservoir [41] and the shear stress could berepeatedly exerted to the nucleic acids. In addition, biomolecules tendto be less stable in liquid form then in dry powder form. Stability is theprime concern in delivering siRNAwith nebulizers. A suitable deliveryvector is therefore required to protect siRNA from both physical andchemical degradation.

Although inhalation is a common way to deliver drug to the lungs,to our best knowledge, none of the in vivo study on siRNA therapyis intended for inhalation. Most of the in vivo studies use eitherintratracheal or intranasal route of delivery to the lungs. This could bedue to the difficulty in formulating inhalable siRNA, especially inmaintaining the stability and biological activity of siRNA duringmanufacturing and delivery process. Recently, Jensen et al. [123]

Table 2Summary of in vivo study of non-viral siRNA delivery to the lung.

Route of administration siRNA/siRNAtarget

Delivery vectors Animal model Notes Year

Ref

Intranasal PAI-1 Naked unmodified siRNA C57BL/6 mice Successfully reduced PAI-1 level inbronchoalveolar fluid

2010[58]

Intranasal siRNA-cy3 Naked unmodified siRNA C57BL/6 mice Low and inhomogeneous siRNA distribution inthe lung

2010[60]

Intranasal GAPDH Polymer (chitosan,chitosan-imidazole)

BALB/c mice ~45% knockdown efficiency for both formulation 2010C57BL/6 mice [99]

Intratracheal siGLO Green Lipid (DharmaFECT) C57BL/6 mice siRNA distributed within epithelium cells ofbronchi and bronchioles

2010

SPARC Successfully reduced inflammation in lungs [59]Intratracheal siRNA-cy3 Naked modified siRNA

(2′O-methyl modification)C57BL/6 mice High and homogenous siRNA distribution in lung 2010

E-cadherin Liposomes (AtuFECT01/DPhyPE/DSPE-PEG)

Naked siRNA produced minor (~21%) knockdownof E-cadherin but not other targets

[60]

VE-cadherin Lipoplex evoked inflammation in lungSFPD

Intratracheal XCL1 Naked unmodified siRNA C57BL/6 mice Expression of XCL1 was suppressed by ~40–50%at mRNA and protein level

2009[36]

Intranasal EHV-1 Naked unmodified siRNA BALB/c mice Successfully inhibit viral infection 2009Lipid (Lipofectamine) No significant difference between the naked

siRNA and lipoplex[31]

Intratracheal siGLO red Liposomes (DOTAP) Athymic nude mice Longer retention in the lungs as compared tointravenous route of administration

2009[100]

Intratracheal EGFP Polymer (PEI and PEI–PEG) C57BL/6 mice ~42% knockdown efficiency of PEI–PEG formulation 2009[70]

Inhalation (Nebulizer) Akt1 Polymer (polyesteramine) A/J mice Successfully suppressed lung cancer progression 2008K-rasLA1 mice [57]

Intratracheal p38 MAP kinase Naked siRNA BALB/c mice Peptide-siRNA formulations did not improveknockdown compared to naked siRNA andinduced inflammatory response

2007

Lipid (cholesterol) Cholesterol-siRNA formulations extends durationbut not magnitude of knockdown compared tonaked siRNA

[61]Cell penetrating peptide(TAT and penetratin)

Intranasal GFP Polymer (chitosan) C57BL/6 mice Significant GFP knockdown in epithelial cells ofbronchioles, ~37% expression of GFP compared toGFP mismatch

2006[101]

Intranasal RSV-P Naked unmodified siRNA(C6-thiol modification)

BALB/c mice Both formulations effectively inhibited RSV infection 2005

Lipid (TransIT-TKO) Transfection efficiency of naked siRNA was ~70–80%of lipoplex

[102]

Intranasal siSC2-5 Naked unmodified siRNA Rhesus macaque Successfully inhibit SCV replication in monkeyrespiratory tract

2005[34]

Intratracheal Fas Naked unmodified siRNA C57BL/6 mice mRNA expression of Fas and caspase 8 weresignificantly reduced in lung tissue

2005

Caspase 8 Animals were protected from hemorrhagicshock and sepsis-induced acute lung injury

[103]

Intratracheal KC Naked unmodified siRNA C57BL/6 mice mRNA expression of KC and MIP-2 were reduced by~40% in lung tissue, IL-6 and MPO activity were alsoreduced

2005MIP-2 [104]

Intranasal HO-1 Naked unmodified siRNA C57BL/6 mice Successfully silence endogenous gene expression inthe lung

2004[105]

Intranasal GAPDH Surfactant (InfaSurf) C57BL/6 mice GAPDH level in lung was inhibited to 50% in 24 h and67% in 7 days

2004[106]

Intranasal+intravenous NP Lipid (Oligofectamine) BLAB/cAnNCR mice Intranasal+intravenous delivery successfullyinhibited viral replication at site of infection, lesseffective when only intravenous route was used

2004PA [107]

6 J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

reported the production of siRNA loaded poly(D,L-lactide-co-glyco-lide) (PLGA) nanoparticles by spray-drying. The integrity andbiological activity of siRNA was successfully preserved during thedrying process and the physicochemical properties of the particles aresuitable for inhalation. It will be interesting to evaluate the in vivoperformance of such formulation.

3.2. Intratracheal route

Intratracheal route is commonly used for lung delivery in animalstudies for all kinds of drugs. However its clinical application is verylimited. The method was initially described as an exposure tech-nique to evaluate respiratory tract toxicity from airborne materials[42]. It was later adapted to assess pulmonary drug delivery, mainlyin rodents [64]. Intratracheal route is a non-physiological and extre-mely uncomfortable delivery technique [42], it is therefore notemployed as a routine route of drug administration for human use.Traditional method requires surgical procedure including tracheot-omy, and the animal has to be anesthetized, usually with sodiumpentobarbital or combination of xylazine and ketamine. The animalis then placed on a surgical board. The trachea is exposed and anendotracheal tube or needle is inserted through an incision madebetween the tracheal cartilaginous rings, projecting its tip at adefined position just before the tracheal bifurcation. The drugsolution or suspension is instilled in the airways through the tubeusing a microsyringe (Fig. 3a).

A relatively non-invasive way to achieve pulmonary delivery viathe intratracheal route was described by Bivas-Benita et al. [124]. Nosurgical procedure is required but the animal still needs to beanesthetized. While still under anesthesia, the tongue of the animal isgently pulled out and a microsprayer or similar device is carefullyinserted endotracheally to deliver the aerosol into the lungs.Alternatively the animal is intubated through the mouth and tracheausing a catheter or needle and the drug is being instilled in solution orsuspension form. In these procedures, since the delivery is madethrough the mouth, it is also referred as oro-tracheal route [64], butcorrect insertion to the trachea may not be straight-forward (Fig. 3b).

Many in vivo studies employed the intratracheal route to deliversiRNA to the lungs [36,59,61,70,100]. The main advantage ofintratracheal delivery is that it ensures high delivery efficiency withminimal drug lost, making it an excellent delivery method for proof-of-concept study when local delivery to the lung is desirable. Thesuccess of intratracheal siRNA delivery to the lungs of mouse was firstreported by Perl et al. [103]. The group demonstrated that by usingmice with over-expression of GFP, the intratracheal instillation ofsiRNA targeting GFP displayed a reduced fluorescent intensity in thelungs but not in the liver or the spleen. To investigate the geneknockdown efficiency, siRNA targeting fas and caspase-8 weredelivered in the same way. The mRNA expression of fas andcaspase-8 in lung tissues was found to be significantly reduced. Thiswork also showed that the siRNAs did not induce lung inflammationas assessed by lung tissue interferon-α, tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 levels. Interestingly, delivery vector was notrequired to achieve in vivo siRNA transfection. Rosas-Taraco et al. [36]also reported successful delivery of naked siRNA through theintratracheal route. After the aerosolized siRNA targeting XCL1 wasadministered intratracheally to mouse infected with tuberculosis, theexpression of XCL1 in the lungs was suppressed by 40–50% at bothmRNA and protein level. Since the expression of XCL1 affects theformation of the lung granuloma which is known to be associatedwith mycobacterium tuberculosis infection, the local delivery ofsiRNA may be exploited as a novel therapy for the treatment oftuberculosis. The observation of successful in vivo gene knockdownmediated by naked siRNA is discussed later.

Some studies were performed to compare siRNA mediatedsilencing with or without delivery vector via the intratracheal route.

Moschos et al. [61] reported that naked siRNA failed to mediate anygene silencing in vitro in mouse fibroblast cell lines whereasconjugation of siRNA to cell penetrating peptides (TAT or penetratin),or lipid carrier (cholesterol) resulted in 20–40% gene silencing effectat mRNA level. Surprisingly when they proceeded to in vivo study,they found that naked siRNA managed to produce a 30–45% geneknockdown at mRNA level. Neither TAT nor penetratin improved thetransfection efficiency as compared of naked siRNA. The use of cellpenetrating peptides was even found to trigger inflammatoryresponse in lung tissues. In a more recent study, Gutbier et al. [60]demonstrated that the epithelial E-cadherin was reduced moderatelyby 21% inmouse lung tissues following intratracheal delivery of nakedtarget-specific siRNA, but no significant reduction of endothelial VE-cadherin or lamin B1 was observed. The use of lipid-based deliveryvector (DPhyPE/DSPE-PEG) was found to evoke inflammatory res-ponse in the lungs.

Despite the success of intratracheal route of siRNA delivery inanimal models, it must be noted that intratracheal route is an artificialway to deliver drug into the lungs. Drug deposition by this route tendsto be less uniform as compared to inhalation [64]. Since intratracheal

Fig. 3. Schematic illustrates the route of siRNAadministration into the lungs used in in vivostudies. (a) Intratracheal route— trachea of the animal is exposed surgically and a tube isinserted through an incisionmade between the tracheal rings. The solution/suspension isinstilled through the tube using a microsyringe. (b) Oro-tracheal route — the animal isintubated from the mouth to the trachea and the solution/suspension is instilled throughthe oral cavity to avoid the need of surgery. (c) Intranasal route — a micropipette orcatheter containing the solution/suspension is inserted gently into the naris of the animaland the solution/suspension is slowly instilled into the nasal cavity.

7J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

route avoids the oropharynx deposition, it reduces drug loss. Inaddition, the effect of aerosol size which is the most critical factoraffecting human lung deposition was not the focus of investigation inthese studies. Therefore it is difficult to rely on this route ofadministration to accurately evaluate the in vivo delivery efficiencyof a particular formulation.

3.3. Intranasal route

Intranasal route is another very popular way to deliver siRNA tothe lungs in animal studies [31,34,58,60,99,101,102,105–107], partlybecause of its easy experimental setup. The animals are usually lightlyto deeply anesthetized. The siRNA formulations are then instilleddrop-wise to the naris to be breathed (Fig. 3c). There were a numberof studies showed success in siRNA delivery to the lung in vivothrough this route. Zhang et al. [105] were the first to demonstratethat lung-specific siRNA delivery could be achieved in mouse byintranasal administration without the need for any delivery vector.Naked siRNA targeting for heme oxygenase-1 (HO-1) effectivelysuppressed the expression of HO-1 gene in the injured lung models ofmouse. Soon, the delivery of naked siRNA via intranasal route wasshown to be successful in inhibiting lung viral infection in mouse[31,102] and rhesus macaque [34].

Since the anatomy and physiology of the lungs between mouseand human are very different, the success we see in intranasal siRNAdelivery to the lungs could not be easily translated to human use.Mice are obligate nasal breathers, it is not surprising to see a highproportion of siRNA delivered through the nose was deposited in thelungs. In addition, the use of anesthetics in these animal studies mayhave impaired the mucociliary action [125], thereby overestimatingthe transfection efficiency of the formulations. In order to evaluate thefeasibility of lung delivery via the nasal route in humans, Heyder et al.[126] performed an experiment on healthy adult volunteers whowere asked to inhale mono-disperse particles through the nose at afixed flow and volume. It was found that only about 3% of 1–5 μmparticles were deposited in the bronchial airways through nosebreathing. The majority of particles were deposited in the noseinstead. For this reason, intranasal route is perfect for delivering siRNAto the nasopharynx area.

In fact one of the human clinical trials, which is currently at phaseII stage, uses this route to deliver siRNA for the treatment of humanRSV infection [15,16]. RSV is an upper respiratory disease. ALN-RSV01is the siRNA, developed by Alnylam Pharmaceuticals, designed toinhibit the replication of RSV by interrupting the synthesis of the viralnucleocapsid protein. Since RSV replicates almost exclusively in thesingle outermost layer of cells of the respiratory epithelium includingthe lining of nasal passages and trachea, local delivery of ALN-RSV01to nasopharynx could potentially treat the infections. ALN-RSV01 isdelivered without a delivery vector as a nasal spray and targets theupper respiratory tract instead of the deep lung area. The author hasalso pointed out that the nasal spray of ALN-RSV01 would not beexpected to reach the lower respiratory tract, and the development ofan aerosolized drug that targets the lower respiratory tract will bedesirable in future trials for the treatment of naturally infectedpatients [16].

Furthermore, intranasal route is being developed for a long time toadminister macromolecules such as proteins and peptides forsystemic delivery [125,127]. The relatively large absorptive surfacearea in the nasal cavity and the high vascularization of the nasalmucosa facilitate rapid absorption. The intranasal route is definitely aroute to explore for systemic delivery of siRNA.

4. Non-viral delivery of siRNA to the lung

A delivery vector is often required to facilitate the cellular deliveryof siRNA as the highly charged, hydrophilic natures of the macro-

molecules make it unable to cross the biological membrane to reachits target sites. Due to the safety concerns with viral-vector, manysiRNA delivery studies focus on the development of non-viral vectors.An ideal siRNA delivery vector should consist of the following criteria:(i) condense siRNA into nanosized particles; (ii) protect siRNA fromenzymatic degradation; (iii) facilitate cellular uptake; (iv) promoteendosomal escape; (v) release siRNA into the cytoplasm where theRISC is located and (vi) negligible toxicity. In addition, optimaldelivery should be achievedwithout compromising the gene silencingactivity and specificity of siRNA. Commonly used non-viral siRNAdelivery vector includes lipids, polymers and peptides. Interestingly,naked siRNA was also found to be successful in producing genesilencing effect in vivo. These vectors are illustrated in Fig. 4 and thepulmonary delivery using each of these systems is discussed in detail.

4.1. Naked siRNA

The term ‘naked siRNA’ or ‘unformulated siRNA’ refers to thedelivery of siRNA without using any delivery agent. This includes thedelivery of both unmodified siRNA and modified siRNA, formulated insaline or other simple excipients such as 5% dextrose. Sinceunmodified siRNA is susceptible to nuclease degradation, chemicallymodified siRNA was introduced initially to address this issue byincreasing the nuclease resistance. The siRNA can also be chemicallymodified to improve potency, increase specificity, reduce immuneresponse and reduce off-target effects. The subject of siRNA modi-fication has been thoroughly reviewed byWatts et al. [128]. Themajorconsideration of siRNA modification is to ensure that the genesilencing efficiency of siRNA is not adversely affected.

While the systemic delivery of naked siRNA generally failed toproduce significant gene silencing effect [35,129,130], surprisinglythere were some successes in delivering naked, unmodified siRNAlocally including to the lung [31,34,36,58,60,102,103,105] (Table 1).Some of these studies have already been discussed in section 2.3.2 and2.3.3. Usually the naked siRNA were administrated either intranasallyor intratracheally to the mouse and the siRNA were targeted againstendogenous or viral genes. In some cases, the use of delivery vectorssuch as lipids or peptides showed no or only marginal improvementof gene silencing efficiency in the lung compared to naked siRNA[31,61,102]. These observations were truly intriguing, as the questionof how the naked siRNA crossed the biological membrane in order toachieve the post-transcriptional gene silencing remains to beanswered, although the reduced nuclease activity in the lung mayprovide part of the explanation [70]. Bitko et al. [102] reported thatnaked unmodified siRNA was able to prevent respiratory viralinfection through siRNA-mediated gene knockdown when thenaked siRNA was administrated intranasally in the mouse. Theauthors suggested that the lung tissue is perhaps more receptive toexchange of molecules naturally or when infected, and this explana-tion is yet to be confirmed.

Nevertheless, the delivery of naked siRNA has been extended toclinical trials. As mentioned in the previous section, ALN-RSV01,which is a modified form of siRNA, were administrated intranasally asnasal spray without the use of any delivery vector and the initial dataconfirmed its efficiency in reducing RSV infection [15,16]. Regardlessof the mystery of how the naked siRNA can cross the cell membrane,gain access into the cytoplasm and remain intact to perform itsbiological action, the performance of naked siRNA so far in animalmodels and human studies are promising. The major advantage ofusing naked siRNA is simplicity without the need to concern about thetoxicity and inflammatory responses associated with certain deliveryvectors. However, it must be stressed that in order to make theformulation applicable for deep lung delivery in human, aerosolinhalation is still the administration route of choice. A particulatecarrier will be required to deliver the siRNA in dry powder form, or adelivery agent will be needed to protect the siRNA from shear force

8 J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

produced during the nebulizing process in suspension form. The useof delivery agents could also enhance specific cells targeting, improvepharmacokinetics and facilitate cellular uptake [131]. There werestudies indicated that the use of optimized delivery vector couldsignificantly improve lung delivery efficiency in animal modelsincluding nonhuman primates as compared to naked siRNA[132,133]. Therefore the development of an effective vector forpulmonary siRNA delivery is still the focus of research in the field.

4.2. Lipid-based delivery vectors

Lipid-based delivery systems are commonly used to deliver siRNAboth in vitro and in vivo [134]. Typically cationic lipids or liposomesare used to form complexes with the negatively charged siRNAthrough spontaneous electrostatic interaction and the complexes arereferred as lipoplexes. Many commercial siRNA transfection agentsare lipids-based system, some of which are also employed for in vivopulmonary delivery, e.g. Oligofectamine™ [107], TransIT-TKO [102]and DharmFECT [59]. The major challenges of using lipid-baseddelivery vectors in the clinic are their toxicity and the non-specificactivation of inflammatory cytokines and interferon responses [135].When the lipoplexes are to be aerosolized, special attention must begiven to the stability [136] as theymay undergo physical and chemicalchanges thatmay result in immature release and hence degradation ofsiRNA.

4.2.1. Cationic lipoplexes and liposomesIn order to maximize stability and delivery efficiency, and

minimize toxic side effects, it is crucial to optimize the lipidcomposition, lipids to siRNA ratio and the lipoplexes preparationmethods. Lipoplexes are easy to prepare and generally have goodtransfection efficiency due to their efficient interaction with thenegatively charged cell membranes. However they also present somedisadvantages such as poor stability and poor reproducibility [137].Moreover, cationic lipids or liposomes are generally more toxic thantheir neutral counterparts. A study performed by Dokka et al. [138]

showed that pulmonary administration of cationic liposomes,Lipofectamine and DOTAP, elicited dose–response toxicity andpulmonary inflammation in mice. The effect was more pronouncedwith the multivalent Lipofectamine than the monovalent DOTAP. Inaddition, they found that neutral and anionic liposomes did notexhibit lung toxicity.

4.2.2. PEGylated lipidsTo circumvent the problem associated with positive surface

charge, hydrophilic polymer such as polyethylene glycol (PEG) hasbeen employed to shield the surface charge of cationic lipids orliposomes, with the attempt to reduce the inflammatory response.PEG covalently linked to a phospholipid has been used for many yearsto prolong circulation in the bloodstream by reducing opsonizationand subsequent cells capture by the mononuclear phagocyte system[139]. The cationic lipid, Genzyme Lipid (GL-67), was previouslyinvestigated for plasmid DNA delivery to the lung for the treatmentcystic fibrosis in human clinical trials [140,141]. GL-67 consisted ofDOPE:DMPE-PEG5000 (at a molar ratio of 1:2:0.05) was complexedwith DNA and the resulting lipoplexes were aerosolized andadministrated to the patients with a jet nebulizer. For patients whoreceived the treatment, their chloride abnormalities were found to besignificantly improved and the bacterial adherence in the lungswas reduced. The promising results have led to the investigation ofthe same lipid-based system for siRNA delivery. Griesenbach et al.[142] assessed the in vivo efficiency of GL-67 in delivering siRNA intothe lung of mice via the intranasal route. The siRNA targetingβ-galactosidase (β-gal) was found to be localized in alveolarmacrophages. β-gal mRNA was reduced by approximately 33%, butthere was no significant change in β-gal protein expression. Perhapsthe use of nebulizer could be considered for further investigation ofthis lipidic system.

4.2.3. Neutral lipidsApart from PEGylation, another way to avoid toxicity and

inflammatory response of lipid-based system is to use neutral lipid-

Fig. 4. Schematic illustration of different non-viral methods commonly used in siRNA delivery.

9J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

based systems. Although neutral systems have the more favorablesafety profile, the lack of interaction with the negatively chargedsiRNA has limited their use as delivery vector. There are severalmethods to overcome this issue. One of the strategies is to link siRNAand lipids together through direct conjugation instead of electrostaticinteraction. It has been demonstrated that chemical conjugation ofsiRNA to cholesterol can facilitate siRNA uptake in vivo after systemicadministration [143]. Moschos et al. [61] applied this strategy topulmonary delivery of siRNA. The cholesterol-siRNA conjugates weredelivered to the mice by intratracheal administration. The duration ofgene knockdown effect was extended by the cholesterol-conjugatedsystem at mRNA level as compared to naked siRNA. However themagnitude of knockdown was not improved. Importantly, thecholesterol-siRNA conjugates did not elicit inflammation in micetissues.

4.2.4. Lipids particlesAn alternative strategy to improve the safety profile of lipid-based

system is to encapsulate the siRNA inside the neutral lipid particles.Semple et al. [144] introduced the utilization of pH sensitive ionizableaminolipids to facilitate efficient encapsulation of antisense oligonu-cleotide in lipid vesicles. The important characteristic of these lipids isthat they exhibit positive charge at acidic pH and neutral charge atphysiological pH, thereby providing means for the lipids to efficientlyinteract with nucleic acids and the lipid vesicles can be renderedneutral at physiological pH. Semple et al. further developed thistechnique to produce stable nucleic acid lipid particles (SNALP) ofuniform size with high siRNA encapsulation and delivery efficiency[145]. The two important parameters underlying lipid design forSNALP-mediated delivery are (i) the pKa of the ionizable cationiclipid, which determines the surface charge of the lipid particles underdifferent pH conditions; and (ii) the ability, when protonated, toinduce a nonbilayer phase structure when mixed with anionic lipids.This property of the lipids determines the membrane destabilizingcapacity and the endosomolytic potential of the lipid particles. TheSNALP demonstrated efficient in vivo gene silencing when adminis-tered intravenously in nonhuman primates [145,146]. These systemscould be further exploited for pulmonary delivery.

4.2.5. Lipid-like moleculesRecently, a new class of lipid-like delivery molecules, termed

lipidoids, was developed for siRNA delivery [132,147]. A combinatoryapproach was employed to allow the rapid synthesis of a large libraryof structurally diverse lipidoids. The library was screened for theability to deliver siRNA in vitro and in vivo. The leading candidate for invivo siRNA mediated gene knockdown was identified as 98N12-5. Thissystem was tested for the ability to inhibit RSV replication in thelungs. After intranasal administration to the mouse model of RSVinfection, the naked siRNA provided one log reduction in viral plaquesin lung tissues, whereas the lipoidoid system at the same dose ofsiRNA provided greater than two log reductions in viral plaques [132].This method provides the possibility of rapid design of new lipid-based materials for pulmonary siRNA delivery.

4.3. Polymer-based delivery vectors

One the attractive properties of polymer-based delivery vectors istheir versatile nature that allows their physicochemical characteristicsto be modified relatively easily to fit their purposes. In addition, it hasbeen suggested that polymers generally do not evoke as strong animmune response as liposomes [13]. In general, polymer-basedvectors can be divided into two categories: polycations and polymericnanoparticles. Synthetic polycations such as polyethylenimine (PEI)[87], polyamidoamine (PAMAM) dendrimers [148] and naturalpolycations such as chitosan [149] are used for delivering DNA for along time. These polymers have high cationic charge density and form

polyplexes spontaneously with the negatively charged nucleic acidsthrough electrostatic interaction. The size of polyplexes is affected bythe molecular weight of polymers, the charge ratio, the pH and ionicstrength of the medium. A net positive charge is required to maintaincolloidal stability of the polyplexes. Because of the difference inphysicochemical properties between plasmid DNA and siRNA, thisapproach is found to be less successful in delivering siRNA [150]. Thestiffer structure of siRNA results in weaker interaction with polyca-tions [151]. As a consequence, the polyplexes are less efficient inprotecting siRNA against nuclease. Increase the amount of polycationsmay offer better protection but it might contribute to toxicity. Thealternative way to deliver siRNA using polymer is to preparepolymeric nanoparticles which are usually solid nanoparticles madefrom hydrophobic polymers such as poly(D,L-lactide-co-glycolide)(PLGA) [122,152]. The siRNA is either dispersed throughout thepolymer matrix, or surrounded by a polymeric shell. The polymericnanoparticles could offer better protection to siRNA, but the loadingefficiency is the main challenge to this delivery method.

4.3.1. PEISynthetic polymer PEI is commonly used in siRNA delivery due to

its high intracellular delivery efficiency. According to the protonsponge hypothesis as mentioned earlier in Section 2.2, the highbuffering capacity of PEI inside the acidic endosomes leads to theswelling and rupture of endosomes, thereby promoting endosomalescape [87]. PEI is considered as the ‘gold standard’ for in vitro genedelivery and its transfection efficiency depends on the molecularweight and degree of branching. Merkel et al. [70] investigated the useof PEI and PEI–PEG polyplexes for siRNA delivery to the lung. Thepolyplexes containing siRNA targeting EGFP were administered to theactin-EGFP expressing mice through intratracheal instillation. Theresult showed that the PEI–PEG system displayed a better deliveryefficiency with a 42% knockdown of EFGP expression in the lungtissues. However the PEI–PEG polyplexes also produced a moderateproinflammatory effect as the levels of TNF-α and IL-6 were elevated.PEI also associates with problems such as relatively high toxicity andthe lack of biodegradability [86]. To tackle these problems, Xu et al.developed a new degradable PEI derivatives, poly(ester amine)-alt-PEG copolymer for in vivo siRNA delivery [57]. A previous studyconfirmed the biodegradability and low toxicity of this polymer [152].For in vivo pulmonary delivery, the polyplexes containing siRNAtargeting Akt1were aerosolized and administered tomousemodels oflung cancer through a nose-only inhalation system. The lung cancerprogression of treated mice was significantly suppressed withoutshowing any significant sign of toxicity. To facilitate rapid develop-ment of PEI derivatives for siRNA delivery, Thomas et al. [153]employed a high throughput synthesis and screening method tocreate a combinatorial library of biodegradable PEI derivatives toidentify a novel vectors for gene delivery. The study focused on DNAdelivery through intravenous injection to the mice and the techniquecould be easily adapted for the investigation of pulmonary siRNAdelivery.

4.3.2. ChitosanChitosan has attracted much attention as potential vector for

siRNA delivery due to its natural, biocompatible, biodegradable andlow toxicity nature. In addition, the mucoadhesive and mucosapermeation properties of chitosan make it particularly favorable forpulmonary delivery. Unfortunately, it only shows moderate in vitroand in vivo transfection efficiency, possibility due to its relativelyweakability to promote endosomal escape [86,149]. Chitosan-basedformulation for siRNA delivery has been thoroughly reviewed byMao et al. recently [154]. The feasibility of using chitosan to deliversiRNA into the lungs was demonstrated by Howard et al. [101].Chitosan/siRNA nanoparticles were prepared by the addition of smallvolume of siRNA into chitosan stock solution in sodium acetate buffer.

10 J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

After the nanoparticles were administered intranasally to EGFP-expressing transgenic mice, the number of EGFP expressing epithelialcells in the bronchioles was reduced by 43% (compare to untreatedcontrol) and 37% (compared to mismatch control) [101], and thestability of the system increased with the molecular weight anddegree of deacetylation [155]. A recent study conducted by the samegroup described the aerosolization of chitosan/siRNA nanoparticlesfor intratracheal administration with the attempt to improve lungdeposition in mice [133]. The aerosol was generated by an AeroP-robe™ nebulizing catheter which was inserted non-invasively intothe animal below the vocal cords. This delivery route was comparedwith the intranasal route and, as expected, the results confirmed thatthe aerosolized formulation improved the lung deposition of the samesiRNA dosage, and the EGFP expression in the transgenic mice modelwas reduced by 68%. However the author also pointed out thatcatheter insertion ensured sufficient delivery and the effect of size inlung deposition was not investigated. The size of aerosol dropletgenerated in this study was around 20 μm which is not suitable forinhalable formulation. It is critical to reduce droplet diameters in therange of 1–5 μm to allow optimal lung deposition in human followinginhalation. To enhance the siRNA delivery efficiency of chitosan, Kataset al. [151] described the ionic gelation method using sodiumtripolyphosphate to produce siRNA nanoparticles. Compare toelectrostatic complexation, the chitosan/siRNA nanoparticles pro-duced by ionic gelationmethod showed a better in vitro gene silencingeffect due to better stability and higher loading efficiency. It would beinteresting to assess the in vivo performance of such system throughpulmonary delivery.

4.3.3. PLGAPLGA is a biodegradable and biocompatible polymer that is being

investigated to provide controlled release of therapeutic macromole-cules such as peptides, proteins and plasmid DNA [156,157]. Theversatility of PLGA allows chemicalmodification andmany derivativesof PLGA with tailored properties are designed to fit their deliverypurposes. Unlike polycations, PLGA does not form polyplexes withnucleic acids. Instead the nucleic acids are encapsulated in the PLGAnanoparticles. By manipulating the degradation rate of PLGA,sustained release of the encapsulated nucleic acids could be achieved.To design an inhalable formulation, particle size is of extremeimportance. Double emulsion-solvent evaporation is conventionallyused to prepare PLGA nanoparticles and the dry particles are thencollected by lyophilization [157]. However large specific surface areaof the particles often leads to aggregation and the ice formation duringlyophilization process also facilitates aggregation. As a result particlesprepared by this method are not desirable for inhalable formulation aslarge particle size is likely to be impacted out in the oropharynx area.Takashima et al. [158] demonstrated that aggregation of PLGAparticles containing plasmid DNA could be effectively minimizedwithout loss of in vitro transfection efficiency using spray-dryingtechnique. This method was soon adapted by Jensen et al. [123] whodemonstrated the production siRNA containing PLGA nanoparticlesby spray-drying with controlled size distribution that are intended forinhalation. PLGA nanoparticles were co-spray-dried with a carbohy-drate excipient such as lactose, trehalose and mannitol to protect thesiRNA containing nanoparticles against the shear forces and raisedtemperature during the process of spray-drying, and to ensure theparticle size is suitable for inhalation. The physicochemical propertiesand the powder yield of the formulation could be optimized byvarying the amount of excipients and the nanoparticles to excipientsratios. The study showed that the integrity and biological activity ofthe siRNA were preserved during the spray-drying process. Thepotential of this technique to generate inhalable siRNA formulationwill be confirmed by further investigation on lung deposition and invivo gene silencing efficiency.

4.4. Peptide-based delivery vectors

Since the discovery of TAT protein fromHIV-1which is responsiblefor the cellular uptake of the virus [159], a variety of cell-penetratingpeptides (CPPs) have been derived or synthesized. CPPs are frequentlyemployed to facilitate the transport of therapeutic macromoleculesinto the cells and this strategy has been extended to the delivery ofsiRNA [160,161]. CPPs and derivatives that are investigated for siRNAdelivery included TAT [61,162,163], penetratin [163–165], transpor-tan [163], MPG [166,167], CADY [168,169] and LAH4 [170]. Thepeptides are either covalently attached to siRNA through disulphidebond formation, or bind to siRNA through electrostatic interaction toform complexes in a non-covalent manner. Due to the sequencediversity of different CPPs, their mechanism of action is also expectedto vary. Some of these peptides improve cellular delivery by efficienttransport of their cargo across the biological membranes, whereasothers promote endosomal escape and prevent lysosomal degrada-tion. However the exact mechanisms of these peptides are stillcontroversial. The activities of different CPPs have been extensivelyreviewed [161,167,171–174].

Although a variety of CPPs have been investigated for siRNAdelivery, very few studies reported their use for in vivo pulmonarydelivery. To date only one group has described the use of CPPs for invivo delivery of siRNA. Using siRNA targeting p38 MAP kinase,Moschos et al. [61,175] compared the gene silencing effect betweennaked siRNA, TAT-siRNA conjugates and penetratin-siRNA conjugatesboth in vitro and in vivo. The peptides were conjugated to the siRNAthrough disulfide linkage. All siRNA conjugates, but not naked siRNA,showed small but significant reduction (20–36%) of p38 MAP kinaseexpression at mRNA level in mouse fibroblast L929 cell lines.Interestingly, when the systems were administrated intratracheallyto mouse model, naked siRNA was able to produce a 30–45%knockdown of p38 MAP kinase in the lung of the animal despite theabsence of knockdown effect in vitro. Although moderate geneknockdown was observed in TAT- and penetratin-siRNA conjugatedsystems, the TAT or penetratin peptide alone also produced similarknockdown level, suggesting that the CPPs could be the modulators ofp38 MAP kinase expression and the gene knockdown effect was notproduced by the siRNA. Furthermore, penetratin-siRNA conjugateswere found to induce an in vivo innate immune response, but not withthe naked siRNA, TAT-siRNA conjugates, penetratin or TAT alone. Theunderstanding of the mechanism of CPPs activity was crucial todetermine their application in siRNA delivery. In addition, it has beensuggested that covalent attachment of CPP to siRNA may have anegative effect on cellular delivery as the biological activity of thepeptides may have been altered during chemical modification [161].The non-covalent complexing method provides an alternativestrategy but the effect in lung delivery to animal model remains tobe seen. More work needs to be done in this area to develop aneffective peptide-based siRNA vector system for in vivo pulmonarydelivery.

5. Conclusions

Since the discovery of siRNA, its therapeutic potential has beenrapidly recognized. Many studies have been carried out in the pastfew years in delivering siRNA to the lungs for the treatment ofvarious lung diseases. However the majority of these investigationsfocus on the design of siRNA molecules to target a specific diseaseinstead of looking into the delivery perspective. In order to makesiRNA therapy practical in treating human lung diseases, we believethat the inhalation route, especially in dry powder form, is the bestof choice. Unfortunately there is very few literature described siRNAformulation specifically designed for inhalation for clinical use. Veryrecently, Jensen et al. [123] reported the spray-drying method toproduce PLGA based siRNA nanoparticles with physiochemical

11J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

properties that are intended for inhalation, but they have notreached the stage of in vivo study yet.

The major challenges to pulmonary delivery of siRNA include: (i)there is no obvious correlation between in vitro and in vivo study (e.g.the naked siRNA failed to show gene silencing effect in vitro in manystudies, but the in vivo studies often proved otherwise); (ii) it is veryhard to translate the information from animal (especially rodentswhich are the most frequently used model) to human as the anatomyand physiology of the respiratory tract between animal and humanare very distinct; and (iii) the administration routes used for animalstudies are not suitable for human use, and it is extremely difficult toevaluate the delivery efficiency of the formulation before it enters theclinical study. Since many studies have successfully proved thetherapeutic efficiency of siRNA on various lung diseases, the finalhurdle we need to overcome is the development of inhalable andstable siRNA formulations for human use in a practical way beforesiRNA therapeutics for lung diseases become available in the clinic.

Acknowledgements

The authors would like to thank The University of Hong Kong, SeedFunding Programme for financial support.

References

[1] P.D. Zamore, RNA interference: listening to the sound of silence, NatureStructural Biology 8 (2001) 746–750.

[2] G.J. Hannon, RNA interference, Nature 418 (2002) 244–251.[3] S.M. Elbashir, W. Lendeckel, T. Tuschl, RNA interference is mediated by 21- and

22-nucleotide RNAs, Genes & Development 15 (2001) 188–200.[4] J.Y. Yu, S.L. DeRuiter, D.L. Turner, RNA interference by expression of short-

interfering RNAs and hairpin RNAs in mammalian cells, Proceedings of theNational Academy of Sciences of the United States of America 99 (2002)6047–6052.

[5] N. Agrawal, P.V.N. Dasaradhi, A. Mohmmed, P. Malhotra, R.K. Bhatnagar, S.K.Mukherjee, RNA interference: biology, mechanism, and applications, Microbi-ology and Molecular Biology Reviews 67 (2003) 657–685.

[6] M.T. McManus, P.A. Sharp, Gene silencing inmammals by small interfering RNAs,Nature Reviews. Genetics 3 (2002) 737–747.

[7] J. Gil, M. Esteban, Induction of apoptosis by the dsRNA-dependent protein kinase(PKR): mechanism of action, Apoptosis 5 (2000) 107–114.

[8] M. Kumar, G.G. Carmichael, Antisense RNA: function and fate of duplex RNA incells of higher eukaryotes, Microbiology and Molecular Biology Reviews 62(1998) 1415–1434.

[9] M.A. McAnuff, G.R. Rettig, K.G. Rice, Potency of siRNA versus shRNA mediatedknockdown in vivo, Journal of Pharmaceutical Sciences 96 (2007) 2922–2930.

[10] A.V. Vlassov, B. Korba, K. Farrar, S. Mukerjee, A.A. Seyhan, H. Ilves, R.L. Kaspar, D.Leake, S.A. Kazakov, B.H. Johnston, shRNAs targeting hepatitis C: effects ofsequence and structural features, and comparison with siRNA, Oligonucleotides17 (2007) 223–236.

[11] P.K. Kaiser, R.C.A. Symons, S.M. Shah, E.J. Quinlan, H. Tabandeh, D.V. Do, G.Reisen, J.A. Lockridge, B. Short, R. Guerciolini, Q.D. Nguyen, I. siRNA-027 study,RNAi-based treatment for neovascular age-related macular degeneration bysiRNA-027, American Journal of Ophthalmology 150 (2010) 33–39.

[12] J. Shen, R. Samul, R.L. Silva, H. Akiyama, H. Liu, Y. Saishin, S.F. Hackett, S. Zinnen,K. Kossen, K. Fosnaugh, C. Vargeese, A. Gomez, K. Bouhana, R. Aitchison, P. Pavco,P.A. Campochiaro, Suppression of ocular neovascularization with siRNA target-ing VEGF receptor 1, Gene Therapy 13 (2006) 225–234.

[13] V. Brower, RNA interference advances to early-stage clinical trials, Journal of theNational Cancer Institute 102 (2010) 1459–1461.

[14] M.E. Davis, J.E. Zuckerman, C.H.J. Choi, D. Seligson, A. Tolcher, C.A. Alabi, Y. Yen,J.D. Heidel, A. Ribas, Evidence of RNAi in humans from systemically administeredsiRNA via targeted nanoparticles, Nature 464 (2010) 1067–U1140.

[15] J. DeVincenzo, J.E. Cehelsky, R. Alvarez, A. Elbashir, S. Elbashir, J. Harborth, I.Toudjarska, L. Nechev, V. Murugaiah, A. Van Vliet, A.K. Vaishnaw, R. Meyers,Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, anovel RNAi antiviral therapeutic directed against respiratory syncytial virus(RSV), Antiviral Research 77 (2008) 225–231.

[16] J. DeVincenzo, R. Lambkin-Williams, T. Wilkinson, J. Cehelsky, S. Nochur, E.Walsh, R. Meyers, J. Gollob, A. Vaishnaw, A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytialvirus, Proceedings of the National Academy of Sciences of the United States ofAmerica 107 (2010) 8800–8805.

[17] N.S. Dejneka, S.H. Wan, O.S. Bond, D.J. Kornbrust, S.J. Reich, Ocular biodistribu-tion of bevasiranib following a single intravitreal injection to rabbit eyes,Molecular Vision 14 (2008) 997–1005.

[18] A.O. Gaber, A. Patel, M.S. Polinsky, E.C. Squiers, Safety and tolerability ofintravenous QPI-1002, a siRNA, for prophylaxis of delayed graft function (DGF)in deceased donor renal allograft recipients at increased risk of DGF: initial

results from a double-blind, phase I first-in-human study, American Journal ofTransplantation 10 (2010) 132-132.

[19] I. Toudjarska, A. Judge, J. Brodsky, K. McClintock, S.D. de Jong, E. Ambegia, T. Buck,A. Akinc, T. Racie, L.B. Jeffs, E. Yaworski, J. Gollob, I. MacLachlan, D.W. Sah, D.Bumcrot, Development of ALN-VSP: an RNAi therapeutic for liver malignancies,Hepatology 50 (2009) 1829.

[20] R. Alvarez, T. Borland, Q. Chen, S. Milstein, T. Nguyen, G. Hinkle, S. Kuchimanchi, J.Costigan, V. Ristoiu, G.Wang, G. Cole, R. Dorkin, A. Akinc, L. Nechev, V. Kosovrasti,L. Tchangov, M. Tracy, L. Jeffs, I. MacLachlan, P. Lutwyche, D. Martins, S. Costelha,M.J. Saraiva, D.W.Y. Sah, ALN-TTR, an RNAi therapeutic for the treatment oftransthyretin-mediated amyloidosis, Amyloid: Journal of Protein FoldingDisorders 17 (2010) 51–52.

[21] S. Costelha, R. Alvarez, T. Borland, L. Nechev, L. Jeffs, D.W.Y. Sah, M.J. Saraiva,Evaluation of ALN-TTR on transthyretin tissue deposition in a FAP V30M animalmodel, Amyloid: Journal of Protein Folding Disorders 17 (2010) 52-52.

[22] S.A. Leachman, R.P. Hickerson, M.E. Schwartz, E.E. Bullough, S.L. Hutcherson, K.M.Boucher, C.D. Hansen, M.J. Eliason, G.S. Srivatsa, D.J. Kornbrust, F.J.D. Smith,W.H.I.McLean, L.M. Milstone, R.L. Kaspar, First-in-human mutation-targeted siRNAphase Ib trial of an inherited skin disorder, Molecular Therapy 18 (2010)442–446.

[23] R.P. Hickerson, F.J.D. Smith, R.E. Reeves, C.H. Contag, D. Leake, S.A. Leachman, L.M.Milstone, W.H.I. McLean, R.L. Kaspar, Single-nucleotide-specific siRNA targetingin a dominant-negative skinmodel, The Journal of Investigative Dermatology 128(2008) 594–605.

[24] M. Aleku, P. Schulz, O. Keil, A. Santel, U. Schaeper, B. Dieckhoff, O. Janke, J.Endruschat, B. Durieux, N. Roder, K. Loffler, C. Lange, M. Fechtner, K. Mopert, G.Fisch, S. Dames, W. Arnold, K. Jochims, K. Giese, B. Wiedenmann, A. Scholz, J.Kaufmann, Atu027, a liposomal small interfering RNA formulation targetingprotein kinase N3, inhibits cancer progression, Cancer Research 68 (2008)9788–9798.

[25] A. Santel, M. Aleku, N. Roder, K. Mopert, B. Durieux, O. Janke, O. Keil, J.Endruschat, S. Dames, C. Lange, M. Eisermann, K. Loffler, M. Fechtner, G. Fisch, C.Vank, U. Schaeper, K. Giese, J. Kaufmann, Atu027 prevents pulmonary metastasisin experimental and spontaneous mouse metastasis models, Clinical CancerResearch 16 (2010) 5469–5480.

[26] S.D. Li, L. Huang, Targeted delivery of antisense oligodeoxynucleotide and smallinterference RNA into lung cancer cells, Molecular Pharmaceutics 3 (2006)579–588.

[27] D. Jere, C.X. Xu, R. Arote, C.H. Yun, M.H. Cho, C.S. Cho, Poly(beta-amino ester) as acarrier for si/shRNA delivery in lung cancer cells, Biomaterials 29 (2008)2535–2547.

[28] A.W. Tong, Small RNAs and non-small cell lung cancer, Current MolecularMedicine 6 (2006) 339–349.

[29] X.L. Ren, Y.M. Xu, W. Bao, H.J. Fu, C.G. Wu, Y. Zhao, Z.K. Li, J. Zhang, S.Q. Li, W.Q.Chen, T. Wang, R. Zhang, L.H. Zhang, G.S. Qian, S.Y. Chen, L.T. Jia, A.G. Yang,Inhibition of non-small cell lung cancer cell proliferation and tumor growth byvector-based small interfering RNAs targeting HER2/neu, Cancer Letters 281(2009) 134–143.

[30] D.E. Zamora-Avila, P. Zapata-Benavides, M.A. Franco-Molina, S. Saavedra-Alonso,L.M. Trejo-Avila, D. Resendez-Perez, J.L. Mendez-Vazquez, J. Isaias-Badillo, C.Rodriguez-Padilla, WT1 gene silencing by aerosol delivery of PEI-RNAicomplexes inhibits B16-F10 lung metastases growth, Cancer Gene Therapy 16(2009) 892–899.

[31] A. Fulton, S.T. Peters, G.A. Perkins, K.W. Jarosinski, A. Damiani, M. Brosnahan, E.L.Buckles, N. Osterrieder, G.R. Van de Walle, Effective treatment of respiratoryalphaherpesvirus infection using RNA interference, PLoS ONE 4 (2009).

[32] R. Alvarez, S. Elbashir, T. Borland, I. Toudjarska, P. Hadwiger, M. John, I. Roehl, S.S.Morskaya, R. Martinello, J. Kahn, M. Van Ranst, R.A. Tripp, J.P. DeVincenzo, R.Pandey, M. Maier, L. Nechev, M. Manoharan, V. Kotelianski, R. Meyers, RNAinterference-mediated silencing of the respiratory syncytial virus nucleocapsiddefines a potent antiviral strategy, Antimicrobial Agents and Chemotherapy 53(2009) 3952–3962.

[33] K. Zhou, H.X. He, Y.Y. Wu, M.X. Duan, RNA interference of avian influenza virusH5N1 by inhibiting viral mRNA with siRNA expression plasmids, Journal ofBiotechnology 135 (2008) 140–144.

[34] B.J. Li, Q.Q. Tang, D. Cheng, C. Qin, F.Y. Xie, Q. Wei, J. Xu, Y.J. Liu, B.J. Zheng, M.C.Woodle, N.S. Zhong, P.Y. Lu, Using siRNA in prophylactic and therapeutic regimensagainst SARS coronavirus in rhesusmacaque, NatureMedicine 11 (2005) 944–951.

[35] Q. Ge, L. Filip, A.L. Bai, T. Nguyen, H.N. Eisen, J. Chen, Inhibition of influenza virusproduction in virus-infectedmice by RNA interference, Proceedings of the NationalAcademy of Sciences of the United States of America 101 (2004) 8676–8681.

[36] A.G. Rosas-Taraco, D.M. Higgins, J. Sanchez-Campillo, E.J. Lee, I.M. Orme, M.Gonzalez-Juarrero, Intrapulmonary delivery of XCL1-targeting small interferingRNA in mice chronically infected with Mycobacterium tuberculosis, AmericanJournal of Respiratory Cell and Molecular Biology 41 (2009) 136–145.

[37] C.C. Lee, B.L. Chiang, RNA interference: new therapeutics in allergic diseases,Current Gene Therapy 8 (2008) 236–246.

[38] R.M. Seguin, N. Ferrari, Emerging oligonucleotide therapies for asthma andchronic obstructive pulmonary disease, Expert Opinion on Investigational Drugs18 (2009) 1505–1517.

[39] M. Ulanova,M.Marcet-Palacios, S.Munoz, S. Asfaha,M.K. Kim, A.D. Schreiber, A.D.Befus, Involvement of Syk kinase in TNF-induced nitric oxide production byairway epithelial cells, Biochemical and Biophysical Research Communications351 (2006) 431–437.

[40] N. Vij, S.Y. Fang, P.L. Zeitlin, Selective inhibition of endoplasmic reticulum-associated degradation rescues Delta F508-cystic fibrosis transmembrane

12 J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

regulator and suppresses interleukin-8 levels, The Journal of BiologicalChemistry 281 (2006) 17369–17378.

[41] R.U. Agu, M.I. Ugwoke, M. Armand, R. Kinget, N. Verbeke, The lung as a route forsystemic delivery of therapeutic proteins and peptides, Respiratory Research 2(2001) 198–209.

[42] K.E. Driscoll, D.L. Costa, G. Hatch, R. Henderson, G. Oberdorster, H. Salem, R.B.Schlesinger, Intratracheal instillation as an exposure technique for theevaluation of respiratory tract toxicity: uses and limitations, ToxicologicalSciences 55 (2000) 24–35.

[43] C.X. Shen, A.K. Buck, X.W. Liu, M. Winkler, S.N. Reske, Gene silencing byadenovirus-delivered siRNA, FEBS Letters 539 (2003) 111–114.

[44] T. Hosono, H. Mizuguchi, K. Katayama, Z.L. Xu, F. Sakurai, A. Ishii-Watabe, K.Kawabata, T. Yamaguchi, S. Nakagawa, T. Mayumi, T. Hayakawa, Adenovirusvector-mediated doxycycline-inducible RNA interference, Human Gene Therapy15 (2004) 813–819.

[45] C.T. Yang, J.M. Li, H.H. Weng, Y.C. Li, H.C. Chen, M.F. Chen, Adenovirus-mediatedtransfer of siRNA against survivin enhances the radiosensitivity of human non-small cell lung cancer cells, Cancer Gene Therapy 17 (2010) 120–130.

[46] Z.C. Han, M. Gorbatyuk, J. Thomas, A.S. Lewin, A. Srivastava, P.W. Stacpoole,Down-regulation of expression of rat pyruvate dehydrogenase E1 alpha gene byself-complementary adeno-associated virus-mediated small interfering RNAdelivery, Mitochondrion 7 (2007) 253–259.

[47] H. Sumimoto, S. Yamagata, A. Shimizu, H. Miyoshi, H. Mizuguchi, T. Hayakawa,M. Miyagishi, K. Taira, Y. Kawakami, Gene therapy for human small-cell lungcarcinoma by inactivation of Skp-2 with virally mediated RNA interference, GeneTherapy 12 (2005) 95–100.

[48] H.Y. Huang, C.C. Lee, B.L. Chiang, Small interfering RNA against interleukin-5decreases airway eosinophilia and hyper-responsiveness, Gene Therapy 15(2008) 660–667.

[49] U. Griesenbach, E. Alton, U.K.C.F.G.T. Co, Gene transfer to the lung: lessonslearned from more than 2 decades of CF gene therapy, Advanced Drug DeliveryReviews 61 (2009) 128–139.

[50] N. Bessis, F.J. GarciaCozar, M.C. Boissier, Immune responses to gene therapyvectors: influence on vector function and effector mechanisms, Gene Therapy 11(2004) S10–S17.

[51] S.E. Raper, N. Chirmule, F.S. Lee, N.A. Wivel, A. Bagg, G.P. Gao, J.M. Wilson, M.L.Batshaw, Fatal systemic inflammatory response syndrome in a ornithinetranscarbamylase deficient patient following adenoviral gene transfer, Molec-ular Genetics and Metabolism 80 (2003) 148–158.

[52] U.P. Dave, N.A. Jenkins, N.G. Copeland, Gene therapy insertional mutagenesisinsights, Science 303 (2004) 333-333.

[53] S. Hacein-Bey-Abina, C. von Kalle, M. Schmidt, F. Le Deist, N.Wulffraat, E. McIntyre,I. Radford, J.L. Villeval, C.C. Fraser, M. Cavazzana-Calvo, A. Fischer, A serious adverseevent after successful gene therapy for X-linked severe combined immunodefi-ciency, The New England Journal of Medicine 348 (2003) 255–256.

[54] D.B. Kohn, M. Sadelain, J.C. Glorioso, Occurrence of leukaemia following genetherapy of X-linked SCID, Nature Reviews. Cancer 3 (2003) 477–488.

[55] E. Marshall, Gene therapy: second child in French trial is found to have leukemia,Science 299 (2003) 320-320.

[56] M. Thomas, J.J. Lu, J.Z. Chen, A.M. Klibanov, Non-viral siRNA delivery to the lung,Advanced Drug Delivery Reviews 59 (2007) 124–133.

[57] C.X. Xu, D. Jere, H. Jin, S.H. Chang, Y.S. Chung, J.Y. Shin, J.E. Kim, S.J. Park, Y.H. Lee,C.H. Chae, K.H. Lee, G.R. Beck, C.S. Cho, M.H. Cho, Poly(ester amine)-mediated,aerosol-delivered Akt1 small interfering RNA suppresses lung tumorigenesis,American Journal of Respiratory and Critical Care Medicine 178 (2008) 60–73.

[58] T. Senoo, N. Hattori, T. Tanimoto, M. Furonaka, N. Ishikawa, K. Fujitaka, Y. Haruta,H. Murai, A. Yokoyama, N. Kohno, Suppression of plasminogen activatorinhibitor-1 by RNA interference attenuates pulmonary fibrosis, Thorax 65(2010) 334–340.

[59] J.C.Wang, S.L. Lai, X.J. Guo, X.F. Zhang, B. de Crombrugghe, S. Sonnylal, F.C. Arnett,X.D. Zhou, Attenuation of fibrosis in vitro and in vivo with SPARC siRNA, ArthritisResearch & Therapy 12 (2010).

[60] B. Gutbier, S.M. Kube, K. Reppe, A. Santel, C. Lange, J. Kaufmann, N. Suttorp, M.Witzenrath, RNAi-mediated suppression of constitutive pulmonary geneexpression by small interfering RNA in mice, Pulmonary Pharmacology &Therapeutics 23 (2010) 334–344.

[61] S.A. Moschos, S.W. Jones, M.M. Perry, A.E. Williams, J.S. Erjefalt, J.J. Turner, P.J.Barnes, B.S. Sproat, M.J. Gait, M.A. Lindsay, Lung delivery studies using siRNAconjugated to TAT(48–60) and penetratin reveal peptide induced reduction ingene expression and induction of innate immunity, Bioconjugate Chemistry 18(2007) 1450–1459.

[62] H. Bisgaard, C. O'Callaghan, G.C. Smaldone, Drug Delivery to the Lung, MarcelDekker, New York, 2002, p. 38.

[63] F. Morén, M.T. Newhouse, M.B. Dolovich, Aerosols in Medicine: Principles,Diagnosis, and Therapy, second ed.Elsevier, Amsterdam, 1993.

[64] M. Sakagami, In vivo, in vitro and ex vivo models to assess pulmonary absorptionand disposition of inhaled therapeutics for systemic delivery, Advanced DrugDelivery Reviews 58 (2006) 1030–1060.

[65] W. Yang, J.I. Peters, R.O. Williams, Inhaled nanoparticles — a current review,International Journal of Pharmaceutics 356 (2008) 239–247.

[66] J. Zhang, L. Wu, H.-K. Chan, W. Watanabe, Formation, characterization, and fateof inhaled drug nanoparticles, Advanced Drug Delivery Reviews 63 (2011)441–455.

[67] N. Sanders, C. Rudolph, K. Braeckmans, S.C. De Smedt, J. Demeester, Extracellularbarriers in respiratory gene therapy, Advanced Drug Delivery Reviews 61 (2009)115–127.

[68] I. Roy, N. Vij, Nanodelivery in airway diseases: challenges and therapeuticapplications, Nanomedicine: Nanotechnology, Biology and Medicine 6 (2010)237–244.

[69] M.R. Knowles, R.C. Boucher, Mucus clearance as a primary innate defensemechanism for mammalian airways, The Journal of Clinical Investigation 109(2002) 571–577.

[70] O.M. Merkel, A. Beyerle, D. Librizzi, A. Pfestroff, T.M. Behr, B. Sproat, P.J. Barth,T. Kissel, Nonviral siRNA delivery to the lung: investigation of PEG–PEIpolyplexes and their in vivo performance, Molecular Pharmaceutics 6 (2009)1246–1260.

[71] J. Roesnecker, S. Naundorf, S.W. Gersting, R.W. Hauck, A. Gessner, P. Nicklaus, R.H.Muller, C. Rudolph, Interaction of bronchoalveolar lavage fluid with polyplexesand lipoplexes: analysing the role of proteins and glycoproteins, The Journal ofGene Medicine 5 (2003) 49–60.

[72] J.E. Duncan, J.A. Whitsett, A.D. Horowitz, Pulmonary surfactant inhibits cationicliposome-mediated gene delivery to respiratory epithelial cells in vitro, HumanGene Therapy 8 (1997) 431–438.

[73] J.S. Patton, Mechanisms of macromolecule absorption by the lungs, AdvancedDrug Delivery Reviews 19 (1996) 3–36.

[74] D.A. Groneberg, P.R. Eynott, S. Lim, T. Oates, R. Wu, I. Carlstedt, P. Roberts, B.McCann, A.G. Nicholson, B.D. Harrison, K.F. Chung, Expression of respiratorymucins in fatal status asthmaticus and mild asthma, Histopathology 40 (2002)367–373.

[75] D.A. Edwards, J. Hanes, G. Caponetti, J. Hrkach, A. BenJebria, M.L. Eskew, J.Mintzes, D. Deaver, N. Lotan, R. Langer, Large porous particles for pulmonarydrug delivery, Science 276 (1997) 1868–1871.

[76] A. Ben-Jebria, M.L. Eskew, D.A. Edwards, Inhalation system for pulmonaryaerosol drug delivery in rodents using large porous particles, Aerosol Science andTechnology 32 (2000) 421–433.

[77] M.M. Arnold, E.M. Gonnan, L.J. Schieber, E.J. Munson, C. Berkland, NanoCiproencapsulation in monodisperse large porous PLGA microparticles, Journal ofControlled Release 121 (2007) 100–109.

[78] S. Ferrari, C. Kitson, R. Farley, R. Steel, C. Marriott, D.A. Parkins, M. Scarpa, B.Wainwright, M.J. Evans, W.H. Colledge, D.M. Geddes, E. Alton, Mucus alteringagents as adjuncts for nonviral gene transfer to airway epithelium, Gene Therapy8 (2001) 1380–1386.

[79] E. Daviskas, S.D. Anderson, J.D. Brannan, H.K. Chan, S. Eberl, G. Bautovich,Inhalation of dry-powder mannitol increases mucociliary clearance, TheEuropean Respiratory Journal 10 (1997) 2449–2454.

[80] E. Daviskas, S.D. Anderson, S. Eberl, I.H. Young, Effect of increasing doses ofmannitol on mucus clearance in patients with bronchiectasis, The EuropeanRespiratory Journal 31 (2008) 765–772.

[81] E. Daviskas, S.D. Anderson, A. Jaques, B. Charlton, Inhaled mannitol improves thehydration and surface properties of sputum in patients with cystic fibrosis, Chest137 (2010) 861–868.

[82] S. Xenariou, U. Griesenbach, S. Ferrari, P. Dean, R.K. Scheule, S.H. Cheng, D.M.Geddes, C. Plank, E. Alton, Using magnetic forces to enhance non-viral genetransfer to airway epithelium in vivo, Gene Therapy 13 (2006) 1545–1552.

[83] P. Dames, B. Gleich, A. Flemmer, K. Hajek, N. Seidl, F. Wiekhorst, D. Eberbeck, I.Bittmann, C. Bergemann, T. Weyh, L. Trahms, J. Rosenecker, C. Rudolph, Targeteddelivery of magnetic aerosol droplets to the lung, Nature Nanotechnology 2(2007) 495–499.

[84] S. Xenariou, U. Griesenbach, H.D. Liang, J. Zhu, R. Farley, L. Somerton, C. Singh, P.K.Jeffery, S. Ferrari, R.K. Scheule, S.H. Cheng, D.M. Geddes, M. Blomley, E. Alton, Useof ultrasound to enhance nonviral lung gene transfer in vivo, Gene Therapy 14(2007) 768–774.

[85] I.A. Khalil, K. Kogure, H. Akita, H. Harashima, Uptake pathways and subsequentintracellular trafficking in nonviral gene delivery, Pharmacological Reviews 58(2006) 32–45.

[86] T. Lebhardt, S. Roesler, M. Beck-Broichsitter, T. Kissel, Polymeric nanocarriers fordrug delivery to the lung, Journal of Drug Delivery Science and Technology 20(2010) 171–180.

[87] O. Boussif, F. Lezoualch, M.A. Zanta, M.D. Mergny, D. Scherman, B.Demeneix, J.P. Behr, A versatile vector for gene and oligonucleotidetransfer into cells in culture and in-vivo polyethylenimine, Proceedingsof the National Academy of Sciences of the United States of America 92(1995) 7297–7301.

[88] H. Hatakeyama, E. Ito, H. Akita, M. Oishi, Y. Nagasaki, S. Futaki, H. Harashima, ApH-sensitive fusogenic peptide facilitates endosomal escape and greatlyenhances the gene silencing of siRNA-containing nanoparticles in vitro and invivo, Journal of Controlled Release 139 (2009) 127–132.

[89] S.W. Choi, S.H. Lee, H. Mok, T.G. Park, Multifunctional siRNA delivery system:polyelectrolyte complex micelles of six-arm PEG conjugate of siRNA and cellpenetrating peptide with crosslinked fusogenic peptide, Biotechnology Progress26 (2010) 57–63.

[90] S.H. Lee, S.H. Kim, T.G. Park, Intracellular siRNA delivery system usingpolyelectrolyte complex micelles prepared from VEGF siRNA-PEG conjugateand cationic fusogenic peptide, Biochemical and Biophysical Research Commu-nications 357 (2007) 511–516.

[91] S. Oliveira, I. van Rooy, O. Kranenburg, G. Storm, R.M. Schiffelers, Fusogenicpeptides enhance endosomal escape improving siRNA-induced silencing ofoncogenes, International Journal of Pharmaceutics 331 (2007) 211–214.

[92] N.R. Yacobi, N. Malmstadt, F. Fazlollahi, L. DeMaio, R. Marchelletta, S.F. Hamm-Alvarez, Z. Borok, K.J. Kim, E.D. Crandall, Mechanisms of alveolar epithelialtranslocation of a defined population of nanoparticles, American Journal ofRespiratory Cell and Molecular Biology 42 (2010) 604–614.

13J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

[93] R.G. Parton, A.A. Richards, Lipid rafts and caveolae as portals for endocytosis:new insights and common mechanisms, Traffic 4 (2003) 724–738.

[94] A.D. Judge, V. Sood, J.R. Shaw, D. Fang, K. McClintock, I. MacLachlan, Sequence-dependent stimulation of the mammalian innate immune response by syntheticsiRNA, Nature Biotechnology 23 (2005) 457–462.

[95] M. Sioud, Innate sensing of self and non-self RNAs by Toll-like receptors, Trendsin Molecular Medicine 12 (2006) 167–176.

[96] M. Sioud, RNA interference and innate immunity, Advanced Drug DeliveryReviews 59 (2007) 153–163.

[97] P.C. Chiang, J.W. Alsup, Y.R. Lai, Y.D. Hu, B.R. Heyde, D. Tung, Evaluation of aerosoldelivery of nanosuspension for pre-clinical pulmonary drug delivery, NanoscaleResearch Letters 4 (2009) 254–261.

[98] A.J. Hickey, L. Garcia-Contreras, Immunological and toxicological implications ofshort-term studies in animals of pharmaceutical aerosol delivery to the lungs:relevance to humans, Critical Reviews in Therapeutic Drug Carrier Systems 18(2001) 387–431.

[99] B. Ghosn, A. Singh, M. Li, A.V. Vlassov, C. Burnett, N. Puri, K. Roy, Efficient genesilencing in lungs and liver using imidazole-modified chitosan as a nanocarrierfor small interfering RNA, Oligonucleotides 20 (2010) 163–172.

[100] O.B. Garbuzenko, M. Saad, S. Betigeri, M. Zhang, A.A. Vetcher, V.A. Soldatenkov, D.C.Reimer, V.P. Pozharov, T. Minko, Intratracheal versus intravenous liposomaldelivery of siRNA, antisense oligonucleotides and anticancer drug, PharmaceuticalResearch 26 (2009) 382–394.

[101] K.A. Howard, U.L. Rahbek, X.D. Liu, C.K. Damgaard, S.Z. Glud, M.O. Andersen, M.B.Hovgaard, A. Schmitz, J.R. Nyengaard, F. Besenbacher, J. Kjems, RNA interferencein vitro and in vivo using a chitosan/siRNA nanoparticle system, MolecularTherapy 14 (2006) 476–484.

[102] V. Bitko, A. Musiyenko, O. Shulyayeva, S. Barik, Inhibition of respiratory virusesby nasally administered siRNA, Nature Medicine 11 (2005) 50–55.

[103] M. Perl, C.S. Chung, J. Lomas-Neira, T.M. Rachel, W.L. Biffl, W.G. Cioffi, A. Ayala,Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonaryapoptosis, inflammation, and neutrophil influx after hemorrhagic shock andsepsis, The American Journal of Pathology 167 (2005) 1545–1559.

[104] J.L. Lomas-Neira, C.S. Chung, D.E. Wesche, M. Perl, A. Ayala, In vivo gene silencing(with siRNA) of pulmonary expression of MIP-2 versus KC results in divergenteffects on hemorrhage-induced, neutrophil-mediated septic acute lung injury,Journal of Leukocyte Biology 77 (2005) 846–853.

[105] X.C. Zhang, P.Y. Shan, D.H. Jiang, P.W. Noble, N.G. Abraham, A. Kappas, P.J. Lee,Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfu-sion-induced lung apoptosis, The Journal of Biological Chemistry 279 (2004)10677–10684.

[106] D. Massaro, G.D. Massaro, L.B. Clerch, Noninvasive delivery of small inhibitoryRNA and other reagents to pulmonary alveoli in mice, American Journal ofPhysiology. Lung Cellular and Molecular Physiology 287 (2004) L1066–L1070.

[107] S.M. Tompkins, C.Y. Lo, T.M. Tumpey, S.L. Epstein, Protection against lethalinfluenza virus challenge by RNA interference in vivo, Proceedings of the NationalAcademy of Sciences of the United States of America 101 (2004) 8682–8686.

[108] L.D. Mastrandrea, T. Quattrin, Clinical evaluation of inhaled insulin, AdvancedDrug Delivery Reviews 58 (2006) 1061–1075.

[109] V. Codrons, F. Vanderbist, R.K. Verbeeck, M. Arras, D. Lison, V. Preat, R. Vanbever,Systemic delivery of parathyroid hormone (1–34) using inhalation dry powdersin rats, Journal of Pharmaceutical Sciences 92 (2003) 938–950.

[110] S.H. Bai, V. Gupta, F. Ahsan, Inhalable lactose-based dry powder formulations oflow molecular weight heparin, Journal of Aerosol Medicine and Pulmonary DrugDelivery 23 (2010) 97–104.

[111] A. Rawat, Q.H. Majumder, F. Ahsan, Inhalable large porous microspheres of lowmolecular weight heparin: in vitro and in vivo evaluation, Journal of ControlledRelease 128 (2008) 224–232.

[112] H.K. Chan, Formulation challenges: protein powders for inhalation, in: M.J.Rathbone (Ed.), Modified-Release Drug Delivery Technology, Informa Health-care, New York, 2008, pp. 867–878.

[113] J. Lenzer, Inhaled insulin is approved in Europe and United States, British MedicalJournal 332 (2006) 321-321.

[114] J. Kling, Inhaled insulin's last gasp? Nature Biotechnology 26 (2008) 479–480.[115] T. Quattrin, A. Belanger, N.J.V. Bohannon, S.L. Schwartz, I.I.I.S.G. Exubera Phase,

Efficacy and safety of inhaled insulin (Exubera) compared with subcutaneousinsulin therapy in patients with type 1 diabetes — results of a 6-month,randomized, comparative trial, Diabetes Care 27 (2004) 2622–2627.

[116] P.A. Hollander, L. Blonde, R. Rowe, A.E. Mehta, J.L. Milburn, K.S. Hershon, J.L.Chiasson, S.R. Levin, I.I.I.S.G. Exubera Phase, Efficacy and safety of inhaled insulin(Exubera) compared with subcutaneous insulin therapy in patients with type 2diabetes— results of a 6-month, randomized, comparative trial, Diabetes Care 27(2004) 2356–2362.

[117] J. Rosenstock, W.T. Cefalu, P.A. Hollander, S.S. Klioze, J. Reis, W.T. Duggan, Safetyand efficacy of inhaled human insulin (Exubera) during discontinuation andreadministration of therapy in adults with type 2 diabetes: a 3-year randomizedcontrolled trial, Diabetes Technology and Therapeutics 11 (2009) 697–705.

[118] V. Alabraba, A. Farnsworth, R. Leigh, P. Dodson, S.C.L. Gough, T. Smyth, Exuberainhaled insulin in patients with type 1 and type 2 diabetes: the first 12 months,Diabetes Technology and Therapeutics 11 (2009) 427–430.

[119] R.A. DeFronzo, R.M. Bergenstal, W.T. Cefalu, J. Pullman, S. Lerman, B.W. Bode, L.S.Phillips, I.I.I.S.G. Exubera Phase, Efficacy of inhaled insulin in patients with type 2diabetes not controlled with diet and exercise — a 12-week, randomized,comparative trial, Diabetes Care 28 (2005) 1922–1928.

[120] S.A. Shoyele, A. Slowey, Prospects of formulating proteins/peptides as aerosols forpulmonary drug delivery, International Journal of Pharmaceutics 314 (2006) 1–8.

[121] M. Ameri, Y.F. Maa, Spray drying of biopharmaceuticals: stability and processconsiderations, Drying Technology 24 (2006) 763–768.

[122] M. Irngartinger, V. Camuglia, M. Damm, J. Goede, H.W. Frijlink, Pulmonarydelivery of therapeutic peptides via dry powder inhalation: effects of micro-nisation and manufacturing, European Journal of Pharmaceutics and Biophar-maceutics 58 (2004) 7–14.

[123] D.M.K. Jensen, D. Cun, M.J. Maltesen, S. Frokjaer, H.M.r. Nielsen, C. Foged, Spraydrying of siRNA-containing PLGA nanoparticles intended for inhalation, Journalof Controlled Release 142 (2010) 138–145.

[124] M. Bivas-Benita, R. Zwier, H.E. Junginger, G. Borchard, Non-invasive pulmonaryaerosol delivery in mice by the endotracheal route, European Journal ofPharmaceutics and Biopharmaceutics 61 (2005) 214–218.

[125] M. Hinchcliffe, L. Illum, Intranasal insulin delivery and therapy, Advanced DrugDelivery Reviews 35 (1999) 199–234.

[126] J. Heyder, J. Gebhart, G. Rudolf, C.F. Schiller, W. Stahlhofen, Deposition ofparticles in the human respiratory tract in the size range 0.005–15 μm, Journal ofAerosol Science 17 (1986) 811–825.

[127] A.A. Hussain, Intranasal drug delivery, Advanced Drug Delivery Reviews 29(1998) 39–49.

[128] J.K. Watts, G.F. Deleavey, M.J. Damha, Chemically modified siRNA: tools andapplications, Drug Discovery Today 13 (2008) 842–855.

[129] B. Urban-Klein, S. Werth, S. Abuharbeid, F. Czubayko, A. Aigner, RNAi-mediatedgene-targeting through systemic application of polyethylenimine (PEI)-com-plexed siRNA in vivo, Gene Therapy 12 (2005) 461–466.

[130] A. Pal, A. Ahmad, S. Khan, I. Sakabe, C.B. Zhang, U.N. Kasid, I. Ahmad, Systemicdelivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1expression and inhibits tumor growth in xenograft model of human prostatecancer, International Journal of Oncology 26 (2005) 1087–1091.

[131] M.A. Behlke, Progress towards in vivo use of siRNAs, Molecular Therapy 13(2006) 644–670.

[132] A. Akinc, A. Zumbuehl, M. Goldberg, E.S. Leshchiner, V. Busini, N. Hossain, S.A.Bacallado, D.N. Nguyen, J. Fuller, R. Alvarez, A. Borodovsky, T. Borland, R.Constien, A. de Fougerolles, J.R. Dorkin, K.N. Jayaprakash, M. Jayaraman, M. John,V. Koteliansky, M. Manoharan, L. Nechev, J. Qin, T. Racie, D. Raitcheva, K.G.Rajeev, D.W.Y. Sah, J. Soutschek, I. Toudjarska, H.P. Vornlocher, T.S. Zimmermann,R. Langer, D.G. Anderson, A combinatorial library of lipid-like materials fordelivery of RNAi therapeutics, Nature Biotechnology 26 (2008) 561–569.

[133] E.J.B. Nielsen, J.M. Nielsen, D. Becker, A. Karlas, H. Prakash, S.Z. Glud, J. Merrison,F. Besenbacher, T.F. Meyer, J. Kjems, K.A. Howard, Pulmonary gene silencing intransgenic EGFP mice using aerosolised chitosan/siRNA nanoparticles, Pharma-ceutical Research 27 (2010) 2520–2527.

[134] Y.C. Tseng, S. Mozumdar, L. Huang, Lipid-based systemic delivery of siRNA,Advanced Drug Delivery Reviews 61 (2009) 721–731.

[135] S.Y. Wu, N.A.J. McMillan, Lipidic systems for in vivo siRNA delivery, The AAPSJournal 11 (2009) 639–652.

[136] M.M. Gaspar, U. Bakowsky, C. Ehrhardt, Inhaled liposomes — current strategiesand future challenges, Journal of Biomedical Nanotechnology 4 (2008) 245–257.

[137] A.R. De Fougerolles, Delivery vehicles for small interfering RNA in vivo, HumanGene Therapy 19 (2008) 125–132.

[138] S. Dokka, D. Toledo, X.G. Shi, V. Castranova, Y. Rojanasakul, Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes, PharmaceuticalResearch 17 (2000) 521–525.

[139] G. Storm, S.O. Belliot, T. Daemen, D.D. Lasic, Surface modifications ofnanoparticles to oppose uptake by the mononuclear phagocyte system,Advanced Drug Delivery Reviews 17 (1995) 31–48.

[140] E. Alton, M. Stern, R. Farley, A. Jaffe, S.L. Chadwick, J. Phillips, J. Davies, S.N. Smith,J. Browning, M.G. Davies, M.E. Hodson, S.R. Durham, D. Li, P.K. Jeffery, M. Scallan,R. Balfour, S.J. Eastman, S.H. Cheng, A.E. Smith, D. Meeker, D.M. Geddes, Cationiclipid-mediated CFTR gene transfer to the lungs and nose of patients with cysticfibrosis: a double-blind placebo-controlled trial, Lancet 353 (1999) 947–954.

[141] F.E. Ruiz, J.P. Clancy, M.A. Perricone, Z. Bebok, J.S. Hong, S.H. Cheng, D.P. Meeker,K.R. Young, R.A. Schoumacher, M.R. Weatherly, L. Wing, J.E. Morris, L. Sindel, M.Rosenberg, F.W. van Ginkel, J.R. McGhee, D. Kelly, R.K. Lyrene, E.J. Sorscher, Aclinical inflammatory syndrome attributable to aerosolized lipid-DNA adminis-tration in cystic fibrosis, Human Gene Therapy 12 (2001) 751–761.

[142] U. Griesenbach, C. Kitson, S.E. Garcia, R. Farley, C. Singh, L. Somerton, H. Painter,R.L. Smith, D.R. Gill, S.C. Hyde, Y.H. Chow, J. Hu, M. Gray, M. Edbrooke, V. Ogilvie,G. MacGregor, R.K. Scheule, S.H. Cheng, N.J. Caplen, E. Alton, Inefficient cationiclipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelialcells in vivo, Respiratory Research 7 (2006).

[143] J. Soutschek, A. Akinc, B. Bramlage, K. Charisse, R. Constien, M. Donoghue, S.Elbashir, A. Geick, P. Hadwiger, J. Harborth, M. John, V. Kesavan, G. Lavine, R.K.Pandey, T. Racie, K.G. Rajeev, I. Rohl, I. Toudjarska, G. Wang, S. Wuschko, D.Bumcrot, V. Koteliansky, S. Limmer, M. Manoharan, H.P. Vornlocher, Therapeuticsilencing of an endogenous gene by systemic administration of modified siRNAs,Nature 432 (2004) 173–178.

[144] S.C. Semple, S.K. Klimuk, T.O. Harasym, N. Dos Santos, S.M. Ansell, K.F. Wong, N.Maurer, H. Stark, P.R. Cullis, M.J. Hope, P. Scherrer, Efficient encapsulation ofantisense oligonucleotides in lipid vesicles using ionizable aminolipids:formation of novel small multilamellar vesicle structures, Biochimica EtBiophysica Acta-Biomembranes 1510 (2001) 152–166.

[145] S.C. Semple, A. Akinc, J.X. Chen, A.P. Sandhu, B.L. Mui, C.K. Cho, D.W.Y. Sah, D.Stebbing, E.J. Crosley, E. Yaworski, I.M. Hafez, J.R. Dorkin, J. Qin, K. Lam, K.G.Rajeev, K.F. Wong, L.B. Jeffs, L. Nechev, M.L. Eisenhardt, M. Jayaraman, M. Kazem,M.A. Maier, M. Srinivasulu, M.J. Weinstein, Q.M. Chen, R. Alvarez, S.A. Barros, S.De, S.K. Klimuk, T. Borland, V. Kosovrasti, W.L. Cantley, Y.K. Tam, M. Manoharan,

14 J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15

Author's personal copy

M.A. Ciufolini, M.A. Tracy, A. de Fougerolles, I. MacLachlan, P.R. Cullis, T.D.Madden, M.J. Hope, Rational design of cationic lipids for siRNA delivery, NatureBiotechnology 28 (2010) 172-U118.

[146] T.S. Zimmermann, A.C.H. Lee, A. Akinc, B. Bramlage, D. Bumcrot, M.N. Fedoruk, J.Harborth, J.A. Heyes, L.B. Jeffs, M. John, A.D. Judge, K. Lam, K. McClintock, L.V.Nechev, L.R. Palmer, T. Racie, I. Rohl, S. Seiffert, S. Shanmugam, V. Sood, J.Soutschek, I. Toudjarska, A.J. Wheat, E. Yaworski, W. Zedalis, V. Koteliansky, M.Manoharan, H.P. Vornlocher, I. MacLachlan, RNAi-mediated gene silencing innon-human primates, Nature 441 (2006) 111–114.

[147] K.T. Love, K.P. Mahon, C.G. Levins, K.A. Whitehead, W. Querbes, J.R. Dorkin, J. Qin,W. Cantley, L.L. Qin, T. Racie, M. Frank-Kamenetsky, K.N. Yip, R. Alvarez, D.W.Y.Sah, A. de Fougerolles, K. Fitzgerald, V. Koteliansky, A. Akinc, R. Langer, D.G.Anderson, Lipid-like materials for low-dose, in vivo gene silencing, Proceedingsof the National Academy of Sciences of the United States of America 107 (2010)1864–1869.

[148] A. Bielinska, J.F. KukowskaLatallo, J. Johnson, D.A. Tomalia, J.R. Baker, Regulationof in vitro gene expression using antisense oligonucleotides or antisenseexpression plasmids transfected using starburst PAMAM dendrimers, NucleicAcids Research 24 (1996) 2176–2182.

[149] W.F. Lai, M.C.M. Lin, Nucleic acid delivery with chitosan and its derivatives,Journal of Controlled Release 134 (2009) 158–168.

[150] D. Cun, L.B. Jensen, H.M. Nielsen, M. Moghimi, C. Foged, Polymeric nanocarriersfor siRNA delivery: challenges and future prospects, Journal of BiomedicalNanotechnology 4 (2008) 258–275.

[151] H. Katas, H.O. Alpar, Development and characterisation of chitosan nanoparticlesfor siRNA delivery, Journal of Controlled Release 115 (2006) 216–225.

[152] M.R. Park, K.O. Han, I.K. Han, M.H. Cho, J.W. Nah, Y.J. Choi, C.S. Cho, Degradablepolyethylenimine-alt-poly(ethylene glycol) copolymers as novel gene carriers,Journal of Controlled Release 105 (2005) 367–380.

[153] M. Thomas, J.J. Lu, C.C. Zhang, J.Z. Chen, A.M. Klibanov, Identification of novelsuperior polycationic vectors for gene delivery by high-throughput synthesisand screening of a combinatorial library, Pharmaceutical Research 24 (2007)1564–1571.

[154] S.R. Mao, W. Sun, T. Kissel, Chitosan-based formulations for delivery of DNA andsiRNA, Advanced Drug Delivery Reviews 62 (2010) 12–27.

[155] X.D. Liu, K.A. Howard, M.D. Dong, M.O. Andersen, U.L. Rahbek, M.G. Johnsen, O.C.Hansen, F. Besenbacher, J. Kjems, The influence of polymeric properties onchitosan/siRNA nanoparticle formulation and gene silencing, Biomaterials 28(2007) 1280–1288.

[156] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug and gene deliveryto cells and tissue, Advanced Drug Delivery Reviews 55 (2003) 329–347.

[157] R.C. Mundargi, V.R. Babu, V. Rangaswamy, P. Patel, T.M. Aminabhavi, Nano/microtechnologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives, Journal of Controlled Release 125 (2008)193–209.

[158] Y. Takashima, R. Saito, A. Nakajima, M. Oda, A. Kimura, T. Kanazawa, H. Okada,Spray-drying preparation of microparticles containing cationic PLGA nano-spheres as gene carriers for avoiding aggregation of nanospheres, InternationalJournal of Pharmaceutics 343 (2007) 262–269.

[159] A.D. Frankel, C.O. Pabo, Cellular uptake of the TAT protein from humanimmunodeficiency virus, Cell 55 (1988) 1189–1193.

[160] A. Eguchi, S.F. Dowdy, siRNA delivery using peptide transduction domains,Trends in Pharmacological Sciences 30 (2009) 341–345.

[161] B.R. Meade, S.F. Dowdy, Exogenous siRNA delivery using peptide transductiondomains/cell penetrating peptides, Advanced Drug Delivery Reviews 59 (2007)134–140.

[162] Y.L. Chiu, A. Ali, C.Y. Chu, H. Cao, T.M. Rana, Visualizing a correlation betweensiRNA localization, cellular uptake, and RNAi in living cells, Chemistry & Biology11 (2004) 1165–1175.

[163] J.J. Turner, S. Jones, M.M. Fabani, G. Ivanova, A.A. Arzumanov, M.J. Gait, RNAtargeting with peptide conjugates of oligonucleotides, siRNA and PNA, BloodCells, Molecules & Diseases 38 (2007) 1–7.

[164] A. Muratovska, M.R. Eccles, Conjugate for efficient delivery of short interferingRNA (siRNA) into mammalian cells, FEBS Letters 558 (2004) 63–68.

[165] T.J. Davidson, S. Harel, V.A. Arboleda, G.F. Prunell, M.L. Shelanski, L.A. Greene, C.M.Troy, Highly efficient small interfering RNA delivery to primary mammalianneurons induces MicroRNA-like effects before mRNA degradation, The Journal ofNeuroscience 24 (2004) 10040–10046.

[166] F. Simeoni, M.C. Morris, F. Heitz, G. Divita, Insight into the mechanism of thepeptide-based gene delivery systemMPG: implications for delivery of siRNA intomammalian cells, Nucleic Acids Research 31 (2003) 2717–2724.

[167] S. Veldhoen, S.D. Laufer, A. Trampe, T. Restle, Cellular delivery of small interferingRNA by a non-covalently attached cell-penetrating peptide: quantitativeanalysis of uptake and biological effect, Nucleic Acids Research 34 (2006)6561–6573.

[168] L. Crombez, G. Aldrian-Herrada, K. Konate, Q.N. Nguyen, G.K. McMaster, R.Brasseur, F. Heitz, G. Divita, A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells, Molecular Therapy17 (2009) 95–103.

[169] K. Konate, L. Crombez, S. Deshayes, M. Decaffmeyer, A. Thomas, R. Brasseur, G.Aldrian, F. Heitz, G. Divita, Insight into the cellular uptake mechanism of asecondary amphipathic cell-penetrating peptide for siRNA delivery, Biochemistry49 (2010) 3393–3402.

[170] B. Langlet-Bertin, C. Leborgne, D. Scherman, B. Bechinger, A.J. Mason, A. Kichler,Design and evaluation of histidine-rich amphipathic peptides for siRNA delivery,Pharmaceutical Research 27 (2010) 1426–1436.

[171] M. Lindgren, M. Hallbrink, A. Prochiantz, U. Langel, Cell-penetrating peptides,Trends in Pharmacological Sciences 21 (2000) 99–103.

[172] H. Brooks, B. Lebleu, E. Vives, Tat peptide-mediated cellular delivery: back tobasics, Advanced Drug Delivery Reviews 57 (2005) 559–577.

[173] S. Futaki, Membrane-permeable arginine-rich peptides and the translocationmechanisms, Advanced Drug Delivery Reviews 57 (2005) 547–558.

[174] S. Deshayes, M.C. Morris, G. Divita, F. Heitz, Cell-penetrating peptides: tools forintracellular delivery of therapeutics, Cellular and Molecular Life Sciences 62(2005) 1839–1849.

[175] S.A. Moschos, A.E. Williams, M.A. Lindsay, Cell-penetrating-peptide-mediatedsiRNA lung delivery, Biochemical Society Transactions 35 (2007) 807–810.

15J.K.W. Lam et al. / Advanced Drug Delivery Reviews 64 (2012) 1–15